Brassica napus seed specific promoters identified by microarray analysis

ABSTRACT

Provided are constructs and methods for expressing a transgene in plant cells and/or plant tissues using gene regulatory elements obtained from  Brassica napus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 15/542,037filed Jul. 6, 2017, which is a national phase entry under 35 U.S.C. §371of international Patent Application PCT/US2015/067584, filed Dec. 28,2015, published in English as International Patent Publication No.WO2016111859 on Jul. 14, 2016, which claims priority to U.S. PatentApplication No. 62/100,389 filed on Jan. 6, 2015, all of which areincorporated in their entirety by reference herein.

TECHNICAL FIELD

This invention is generally related to the field of plant molecularbiology, and more specifically, to the field of expression of transgenesin plants.

BACKGROUND

Many plant species are capable of being transformed with transgenes tointroduce agronomically desirable traits or characteristics. Theresulting transgenic plant species are developed and/or modified to haveparticular desirable traits. Generally, desirable traits include, forexample, improving nutritional value quality, increasing yield,conferring pest or disease resistance, increasing drought and stresstolerance, improving horticultural qualities (e.g., pigmentation andgrowth), imparting herbicide tolerance, enabling the production ofindustrially useful compounds and/or materials from the plant, and/orenabling the production of pharmaceuticals.

Transgenic plant species comprising multiple transgenes stacked at asingle genomic locus are produced via plant transformation technologies.Plant transformation technologies result in the introduction of atransgene into a plant cell, recovery of a fertile transgenic plant thatcontains the stably integrated copy of the transgene in the plantgenome, and subsequent transgene expression via transcription andtranslation of the plant genome results in transgenic plants thatpossess desirable traits and phenotypes. However, mechanisms that allowthe production of transgenic plant species to highly express multipletransgenes engineered as a trait stack are desirable.

Likewise, mechanisms that allow the expression of a transgene withinparticular tissues or organs of a plant are desirable. For example,increased resistance of a plant to infection by soil-borne pathogensmight be accomplished by transforming the plant genome with apathogen-resistance gene such that pathogen-resistance protein isrobustly expressed within the roots of the plant. Alternatively, it maybe desirable to express a transgene in plant tissues that are in aparticular growth or developmental phase such as, for example, celldivision or elongation. Furthermore, it may be desirable to express atransgene in leaf and stem tissues of a plant.

Described herein are Brassica napus GALE1 gene promoter regulatoryelements, constructs/vectors containing the Brassica napus GALE1 genepromoter regulatory, and methods utilizing Brassica napus GALE1 genepromoter regulatory elements.

DISCLOSURE

Disclosed herein are sequences, constructs, and methods for expressing atransgene in plant cells and/or plant tissues. In an embodiment thedisclosure relates to a gene expression cassette comprising a promoteroperably linked to a transgene, wherein the promoter comprises apolynucleotide that hybridizes under stringent conditions to apolynucleotide probe comprising a sequence identity of at least 90% to acomplement of SEQ ID NO:1. In further embodiments, the promotercomprises a polynucleotide that has at least 90% sequence identity toSEQ ID NO:1. In additional embodiments, the promoter comprises apolynucleotide comprising an intron. In other embodiments, the intronhas at least 90% sequence identity to a rice actin intron, a maizeubiquitin intron, or an Arabadiopsis thaliana ubiquitin 10 intron. In anembodiment the promoter comprises a polynucleotide comprising a5′-untranslated region. In other embodiments, the operably linkedtransgene encodes a polypeptide or a small RNA. In a subsequentembodiment, the transgene is selected from the group consisting ofinsecticidal resistance transgene, herbicide tolerance transgene,nitrogen use efficiency transgene, water use efficiency transgene,nutritional quality transgene, DNA binding transgene, and selectablemarker transgene. In yet another embodiment, the gene expressioncassette further comprises a 3′-untranslated region. In an embodimentthe 3′-untranslated region comprises a polynucleotide that has asequence identity of at least 90% to SEQ ID NO:2. In an embodiment, arecombinant vector comprises the gene expression cassette. In a furtheraspect of the embodiment, the recombinant vector is selected from thegroup consisting of a plasmid, a cosmid, a bacterial artificialchromosome, a virus, and a bacteriophage. In an embodiment, a transgeniccell comprises the gene expression cassette. In a subsequent aspect ofthe embodiment, the cell is a transgenic plant cell. In an embodiment, atransgenic plant comprises the transgenic plant cell. In a furtheraspect of the embodiment, the transgenic plant is a monocotyledonousplant or dicotyledonous plant. In other aspects of the embodiment, thedicotyledonous plant is selected from the group consisting of anArabidopsis plant, a tobacco plant, a soybean plant, a canola plant anda cotton plant. In an embodiment, a transgenic seed is obtained from thetransgenic plant. In a subsequent embodiment, the promoter is atissue-preferred promoter. In an additional embodiment, thetissue-preferred promoter is an ovule or seed tissue-preferred promoter.In an embodiment, the seed tissue-preferred promoter is an endospermtissue-preferred promoter. In yet another embodiment, the promotercomprises a polynucleotide sequence of nucleotides 1-1429 of SEQ IDNO:1.

In an embodiment the disclosure relates to a transgenic cell comprisinga synthetic polynucleotide that hybridizes under stringent conditions toa polynucleotide probe comprising a sequence identity of at least 90% toa complement of SEQ ID NO:1. In an additional embodiment, the syntheticpolynucleotide has at least 90% sequence identity to SEQ ID NO:1. Inadditional embodiments, the synthetic polynucleotide comprises apolynucleotide comprising an intron. In other embodiments, the intronhas a sequence identity of at least 90% to a rice actin intron, a maizeubiquitin intron, or an Arabadiopsis thaliana ubiquitin 10 intron. In anembodiment, the synthetic polynucleotide comprises a 5′-untranslatedregion. In a further embodiment, the transgenic cell is a transgenicplant cell. In a subsequent embodiment, the transgenic plant cell isproduced by a plant transformation method. In an additional embodiment,the plant transformation method is selected from the group consisting ofan Agrobacterium-mediated transformation method, a biolisticstransformation method, a silicon carbide transformation method, aprotoplast transformation method, and a liposome transformation method.In an embodiment, a transgenic plant comprises the transgenic plantcell. In a further embodiment, the transgenic plant is amonocotyledonous plant or dicotyledonous plant. In other embodiments,the monocotyledonous plant is selected from the group consisting of amaize plant, a rice plant, and a wheat plant. In other aspects of theembodiment, the dicotyledonous plant is selected from the groupconsisting of an Arabidopsis plant, a tobacco plant, a soybean plant, acanola plant and a cotton plant. In an embodiment, a transgenic seed isobtained from the transgenic plant. In an additional embodiment, thepromoter is a tissue-preferred promoter. In an additional embodiment,the tissue-preferred promoter is an ovule or seed tissue-preferredpromoter. In an embodiment, the seed tissue-preferred promoter is anendosperm tissue-preferred promoter. In another embodiment, thesynthetic polynucleotide comprises a polynucleotide sequence ofnucleotides 1-1429 of SEQ ID NO:1.

In an embodiment the disclosure relates to a purified polynucleotidepromoter, wherein the promoter comprises a polynucleotide thathybridizes under stringent conditions to a polynucleotide probecomprising a sequence identity of at least 90% to a complement of SEQ IDNO:1. In further embodiments, the purified polynucleotide promoter hasat least 90% sequence identity to SEQ ID NO:1. In additionalembodiments, the purified polynucleotide promoter comprises apolynucleotide comprising an intron. In other embodiments, the intronhas at least 90% identity to a rice actin intron, a maize ubiquitinintron, or an Arabadiopsis thaliana ubiquitin 10 intron. In anembodiment, the purified polynucleotide promoter comprises a5′-untranslated region. In another embodiment, the purifiedpolynucleotide is operably linked to a transgene. In a subsequentembodiment, the operably linked transgene encodes a polypeptide or is asmall RNA. In an embodiment, a gene expression cassette comprises thepurified polynucleotide sequence operably linked to the transgene, whichis operably linked to a 3′-untranslated region. In an embodiment the3′-untranslated region comprises a polynucleotide that has a sequenceidentity of at least 90% to SEQ ID NO:2. In another embodiment, thetransgene is selected from the group consisting of insecticidalresistance transgene, herbicide tolerance transgene, nitrogen useefficiency transgene, water use efficiency transgene, nutritionalquality transgene, DNA binding transgene, and selectable markertransgene. In an embodiment, a recombinant vector comprises the geneexpression cassette. In an additional embodiment, the recombinant vectoris selected from the group consisting of a plasmid vector, a cosmidvector, and a BAC vector. In an embodiment, a transgenic cell comprisesthe gene expression cassette. In a subsequent embodiment the transgeniccell is a transgenic plant cell. In an embodiment, a transgenic plantcomprises the transgenic plant cell. In an additional embodiment, thetransgenic plant is a monocotyledonous or dicotyledonous plant. In yet afurther embodiment, the monocotyledonous plant is selected from thegroup consisting of a maize plant, a wheat plant, and a rice plant. Inother aspects of the embodiment, the dicotyledonous plant is selectedfrom the group consisting of an Arabidopsis plant, a tobacco plant, asoybean plant, a canola plant and a cotton plant. In an embodiment, atransgenic seed is obtained from the transgenic plant. In a subsequentembodiment, the purified polynucleotide sequence promotestissue-preferred expression of a transgene. In an additional embodiment,the tissue-preferred promoter is an ovule or seed tissue-preferredpromoter. In an embodiment, the seed tissue-preferred promoter is anendosperm tissue-preferred promoter. In other embodiments, the purifiedpolynucleotide comprises a polynucleotide sequence of nucleotides 1-1429of SEQ ID NO:1.

In an embodiment the disclosure relates to a method for expressing aheterologous coding sequence in a transgenic plant, the methodcomprising:

-   -   a) transforming a plant cell with a gene expression cassette        comprising a polynucleotide sequence comprising a sequence        identity of at least 90% to SEQ ID NO:1 operably linked to the        heterologous coding sequence, which is operably linked to a        3′-untranslated region;    -   b) isolating the transformed plant cell comprising the gene        expression cassette;    -   c) regenerating the transformed plant cell into a transgenic        plant; and,    -   d) obtaining the transgenic plant, wherein the transgenic plant        comprises the gene expression cassette comprising the        polynucleotide sequence comprising SEQ ID NO:1.

In additional embodiments, the polynucleotide sequence comprises anintron. In other embodiments, the intron has a sequence identity of atleast 90% to a rice actin intron, a maize ubiquitin intron, or anArabadiopsis thaliana ubiquitin 10 intron. In an embodiment, thepolynucleotide sequence has at least 90% sequence identity to SEQ IDNO:1. In a further embodiment, the heterologous coding sequence isselected from the group consisting of insecticidal resistance codingsequences, herbicide tolerance coding sequences, nitrogen use efficiencycoding sequences, water use efficiency coding sequences, nutritionalquality coding sequences, DNA binding coding sequences, and selectablemarker coding sequences. In an additional embodiment, transforming of aplant cell utilizes a plant transformation method. In yet anotherembodiment, the plant transformation method is selected from the groupconsisting of an Agrobacterium-mediated transformation method, abiolistics transformation method, a silicon carbide transformationmethod, a protoplast transformation method, and a liposometransformation method. In other embodiments, the transgenic plant is amonocotyledonous transgenic plant or a dicotyledonous transgenic plant.In further embodiments, the monocotyledonous transgenic plant isselected from the group consisting of a maize plant, a wheat plant, anda rice plant. In an embodiment, a transgenic seed is obtained from thetransgenic plant. In other aspects of the embodiment, the dicotyledonousplant is selected from the group consisting of an Arabidopsis plant, atobacco plant, a soybean plant, a canola plant and a cotton plant. In afurther embodiment, the heterologous coding sequence is preferentiallyexpressed in a tissue. In an additional embodiment, the tissue-preferredpromoter is an ovule or seed tissue-preferred promoter. In anembodiment, the seed tissue-preferred promoter is an endospermtissue-preferred promoter. In other embodiments, the polynucleotidecomprises a sequence of nucleotides 1-1429 of SEQ ID NO:1.

In an embodiment the disclosure relates to a method for isolating apolynucleotide sequence comprising a sequence identity of at least 90%to SEQ ID NO:1, the method comprising:

-   -   a) identifying the polynucleotide sequence comprising a sequence        identity of at least 90% to SEQ ID NO:1;    -   b) producing a plurality of oligonucleotide primer sequences,        wherein the oligonucleotide primer sequences bind to the        polynucleotide sequence comprising a sequence identity of at        least 90% to SEQ ID NO:1;    -   c) amplifying the polynucleotide sequence comprising a sequence        identity of at least 90% to SEQ ID NO:1 from a DNA sample with        oligonucleotide primer sequences selected from the plurality of        oligonucleotide primer sequences; and,    -   d) isolating the polynucleotide sequence comprising a sequence        identity of at least 90% to SEQ ID NO:1.

In additional embodiments, the polynucleotide sequence comprises anintron. In other embodiments, the intron has a sequence identity of atleast 90% to a rice actin intron, a maize ubiquitin intron, or anArabadiopsis thaliana ubiquitin 10 intron. In an embodiment, thepolynucleotide sequence comprise a 5′-untranslated region. In anadditional embodiment, the isolated polynucleotide sequence comprising asequence identity of at least 90% to SEQ ID NO:1 is operably linked to atransgene. In a further embodiment, the operably linked transgeneencodes a polypeptide. In an embodiment, a gene expression cassettecomprises a polynucleotide sequence with at least 90% sequence identityto SEQ ID NO:1 operably linked to a transgene, wherein the transgene isoperably linked to a 3′-untranslated region. In an embodiment the3′-untranslated region comprises a polynucleotide that has a sequenceidentity of at least 90% to SEQ ID NO:2. In a further embodiment, thetransgene is selected from the group consisting of insecticidalresistance coding sequences, herbicide tolerance coding sequences,nitrogen use efficiency coding sequences, water use efficiency codingsequences, nutritional quality coding sequences, DNA binding codingsequences, and selectable marker coding sequences. In an embodiment, arecombinant vector comprises the gene expression cassette. In a furtherembodiment, the vector is selected from the group consisting of aplasmid vector, a cosmid vector, and a BAC vector. In an embodiment, atransgenic cell comprises the gene expression cassette. In an additionalembodiment, the transgenic cell is a transgenic plant cell. In anembodiment, a transgenic plant comprises the transgenic plant cell. Inan additional embodiment, the transgenic plant is a monocotyledonousplant or a dicotyledonous plant. In a further embodiment, themonocotyledonous plant is selected from the group consisting of a maizeplant, a wheat plant, and a rice plant. In an embodiment, a transgenicseed is obtained from the transgenic plant. In other aspects of theembodiment, the dicotyledonous plant is selected from the groupconsisting of an Arabidopsis plant, a tobacco plant, a soybean plant, acanola plant and a cotton plant. In other embodiments, the isolatedpolynucleotide comprises a polynucleotide sequence of nucleotides 1-1429of SEQ ID NO:1.

In an embodiment the disclosure relates to a method for manufacturing asynthetic polynucleotide sequence comprising a sequence identity of atleast 90% to SEQ ID NO:1, the method comprising:

-   -   a) identifying the polynucleotide sequence comprising SEQ ID        NO:1;    -   b) isolating the polynucleotide sequence comprising SEQ ID NO:1;    -   c) defining a plurality of polynucleotide sequences that        comprise a sequence identity of at least 90% to SEQ ID NO:1;    -   d) synthesizing a polynucleotide sequence comprising a sequence        identity of at least 90% to SEQ ID NO:1; and,    -   e) manufacturing a synthetic polynucleotide sequence comprising        a sequence identity of at least 90% to SEQ ID NO:1.

In a further embodiment, the synthesizing comprises:

-   -   a) identifying the polynucleotide sequence comprising a sequence        identity of at least 90% to SEQ ID NO:1;    -   b) producing a plurality of oligonucleotide primer sequences,        wherein the oligonucleotide primer sequences bind to the        polynucleotide sequence comprising a sequence identity of at        least 90% to SEQ ID NO:1;    -   c) ligating the plurality of oligonucleotide primer sequences to        synthesize the polynucleotide sequence comprising a sequence        identity of at least 90% to SEQ ID NO:1.

In additional embodiments, the synthesized polynucleotide sequencecomprises an intron. In other embodiments, the intron has a sequenceidentity of at least 90% to a rice actin intron, a maize ubiquitinintron, or an Arabadiopsis thaliana ubiquitin 10 intron. In anembodiment, the synthesized polynucleotide sequence comprises a5′-untranslated region. In an additional embodiment, the synthesizedpolynucleotide sequence comprises a sequence identity of at least 90% toSEQ ID NO:1 that is operably linked to a transgene. In yet anotherembodiment, the operably linked transgene encodes a polypeptide. In anembodiment, a gene expression cassette comprises the synthesizedpolynucleotide sequence comprising a sequence identity of at least 90%to SEQ ID NO:1 operably linked to the transgene, that is operably linkedto a 3′-untranslated region. In an embodiment the 3′-untranslated regioncomprises a polynucleotide that has a sequence identity of at least 90%to SEQ ID NO:2. In yet another embodiment, the transgene is selectedfrom the group consisting of insecticidal resistance transgene,herbicide tolerance transgene, nitrogen use efficiency transgene, wateruse efficiency transgene, nutritional quality transgene, DNA bindingtransgene, and selectable marker transgene. In an embodiment, arecombinant vector comprises the gene expression cassette. In anadditional embodiment, the recombinant vector is selected from the groupconsisting of a plasmid vector, a cosmid vector, and a BAC vector. In anembodiment, a transgenic cell comprises the gene expression cassette. Ina further embodiment, the transgenic cell is a transgenic plant cell. Inan embodiment, a transgenic plant comprises the transgenic plant cell.In a further embodiment, the transgenic plant is a monocotyledonous ordicotyledonous plant. In other embodiments, the monocotyledonous plantis selected from the group consisting of a maize plant, a wheat plantand a rice plant. In other aspects of the embodiment, the dicotyledonousplant is selected from the group consisting of an Arabidopsis plant, atobacco plant, a soybean plant, a canola plant and a cotton plant. In anembodiment, a transgenic seed is obtained from the transgenic plant. Inother embodiments, the synthetic polynucleotide comprises apolynucleotide sequence of nucleotides 1-1429 of SEQ ID NO:1.

In an embodiment, a construct includes a gene expression cassettecomprising a Brassica napus GALE1 gene promoter of SEQ ID NO:1. In anembodiment, a gene expression cassette includes a Brassica napus GALE1gene promoter of SEQ ID NO:1 operably linked to a transgene or aheterologous coding sequence. In an embodiment, a gene expressioncassette includes a Brassica napus GALE1 gene 3′-UTR of SEQ ID NO:2operably linked to a transgene. In an embodiment, a gene expressioncassette includes a Brassica napus GALE1 gene 3′-UTR of SEQ ID NO:2operably linked to a promoter. In a further embodiment, a geneexpression cassette includes a Brassica napus GALE1 gene 3′-UTR of SEQID NO:2 operably linked to a Brassica napus GALE1 gene promoter of SEQID NO:1. In an embodiment, a gene expression cassette includes aBrassica napus GALE1 gene promoter of SEQ ID NO:1 operably linked to atransgene or a heterologous coding sequence. In an embodiment, a geneexpression cassette includes at least one, two, three, four, five, six,seven, eight, nine, ten, or more transgenes.

In an embodiment, a gene expression cassette includes independently a) aBrassica napus GALE1 gene promoter of SEQ ID NO:1, and b) a Brassicanapus GALE1 gene 3′-UTR of SEQ ID NO:2.

Methods of growing plants expressing a transgene using Brassica napusGALE1 gene promoters of SEQ ID NO:1, and 3′-UTRs of SEQ ID NO:2 aredisclosed herein. Methods of culturing plant tissues and cellsexpressing a transgene using the Brassica napus GALE1 gene promoters ofSEQ ID NO:1, and 3′-UTRs of SEQ ID NO:2 are also disclosed herein. In anembodiment, methods, as disclosed herein, include tissue-specific geneexpression in plant leaves and stems.

In an embodiment, a gene expression cassette includes a promoterpolynucleotide sequence of SEQ ID NO:1 that was obtained from theBrassica napus GALE1 gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plasmid map of pDAB113903.

FIG. 2 shows a plasmid map of pDAB9381.

FIG. 3 is a microscopy image of Yellow Fluorescent Protein expressionpatterns in transgenic Arabidopsis plant tissues. The image provided inPanel D shows early ovule expression that was localized in the endospermfor the YFP protein, driven by the Brassica napus GALE1 promoter andterminated by 3′ UTR regulatory elements as described in pDAB113903. Theimage provided in Panel E shows expression of the YFP protein, driven bythe Brassica napus GALE1 promoter and terminated by 3′ UTR regulatoryelements as described in pDAB113903, in other plant tissues such asroots, petioles and flowers. The expression of the YFP protein in theroot, petiole and flower tissue was observed in multi-copy numberevents.

MODE(S) FOR CARRYING OUT THE INVENTION Definitions

As used herein, the articles, “a,” “an,” and “the” include pluralreferences unless the context clearly and unambiguously dictatesotherwise.

As used herein, the term “backcrossing” refers to a process in which abreeder crosses hybrid progeny back to one of the parents, for example,a first generation hybrid F1 with one of the parental genotypes of theF1 hybrid.

As used herein, the term “intron” refers to any nucleic acid sequencecomprised in a gene (or expressed nucleotide sequence of interest) thatis transcribed but not translated. Introns include untranslated nucleicacid sequence within an expressed sequence of DNA, as well ascorresponding sequence in RNA molecules transcribed therefrom.

A construct described herein can also contain sequences that enhancetranslation and/or mRNA stability such as introns. An example of onesuch intron is the first intron of gene II of the histone variant ofArabidopsis thaliana or any other commonly known intron sequence.Introns can be used in combination with a promoter sequence to enhancetranslation and/or mRNA stability.

As used herein, the terms “5′ untranslated region” or “5′-UTR” refer toan untranslated segment in the 5′ terminus of pre-mRNAs or mature mRNAs.For example, on mature mRNAs, a 5′-UTR typically harbors on its 5′ end a7-methylguanosine cap and is involved in many processes such assplicing, polyadenylation, mRNA export towards the cytoplasm,identification of the 5′ end of the mRNA by the translational machinery,and protection of the mRNAs against degradation.

As used herein, the term “3′ untranslated region” or “3′-UTR” refers toan untranslated segment in a 3′ terminus of the pre-mRNAs or maturemRNAs. For example, on mature mRNAs this region harbors the poly-(A)tail and is known to have many roles in mRNA stability, translationinitiation, and mRNA export.

As used herein, the term “polyadenylation signal” refers to a nucleicacid sequence present in mRNA transcripts that allows for transcripts,when in the presence of a poly-(A) polymerase, to be polyadenylated onthe polyadenylation site, for example, located 10 to 30 bases downstreamof the poly-(A) signal. Many polyadenylation signals are known in theart and are useful for the present invention. An exemplary sequenceincludes AAUAAA and variants thereof, as described in Loke J., et al.,(2005) Plant Physiology 138(3); 1457-1468.

As used herein, the term “isolated” refers to a biological component(including a nucleic acid or protein) that has been separated from otherbiological components in the cell of the organism in which the componentnaturally occurs (i.e., other chromosomal and extra-chromosomal DNA).

As used herein, the term “purified” in reference to nucleic acidmolecules does not require absolute purity (such as a homogeneouspreparation); instead, it represents an indication that the sequence isrelatively more pure than in its native cellular environment (comparedto the natural level this level should be at least 2-5 fold greater,e.g., in terms of concentration or gene expression levels). The DNAmolecules may be obtained directly from total genomic DNA or from totalgenomic RNA. In addition, cDNA clones are not naturally occurring, butrather are preferably obtained via manipulation of a partially purified,naturally occurring substance (messenger RNA that is reverse transcribedby a reverse transcriptase polymerase). The construction of a cDNAlibrary from mRNA involves the creation of a synthetic substance (cDNA).Individual cDNA clones can be purified from the synthetic library byclonal selection of the cells carrying the cDNA library. Thus, theprocess which includes the construction of a cDNA library from mRNA andpurification of distinct cDNA clones yields an approximately 10⁶-foldpurification of the native message. Likewise, a promoter DNA sequencecould be cloned into a plasmid. Such a clone is not naturally occurring,but rather is preferably obtained via manipulation of a partiallypurified, naturally occurring substance such as a genomic DNA library ordirectly from genomic DNA. Thus, purification of at least one order ofmagnitude, preferably two or three orders, and more preferably four orfive orders of magnitude is favored in these techniques.

Similarly, purification represents an indication that a chemical orfunctional change in the component DNA sequence has occurred. Nucleicacid molecules and proteins that have been “purified” include nucleicacid molecules and proteins purified by standard purification methods.The term “purified” also embraces nucleic acids and proteins prepared byrecombinant DNA methods in a host cell (e.g., plant cells), as well aschemically-synthesized nucleic acid molecules, proteins, and peptides.

The term “recombinant” refers to a cell or organism in which geneticrecombination has occurred. It also includes a molecule (e.g., a vector,plasmid, nucleic acid, polypeptide, or a small RNA) that has beenartificially or synthetically (i.e., non-naturally) altered by humanintervention. The alteration can be performed on the molecule within, orremoved from, its natural environment or state.

As used herein, the term “expression” refers to the process by which apolynucleotide is transcribed into mRNA (including small RNA molecules)and/or the process by which the transcribed mRNA (also referred to as“transcript”) is subsequently translated into peptides, polypeptides, orproteins. Gene expression can be influenced by external signals, forexample, exposure of a cell, tissue, or organism to an agent thatincreases or decreases gene expression. Expression of a gene can also beregulated anywhere in the pathway from DNA to RNA to protein. Regulationof gene expression occurs, for example, through controls acting ontranscription, translation, RNA transport and processing, degradation ofintermediary molecules such as mRNA, or through activation,inactivation, compartmentalization, or degradation of specific proteinmolecules after they have been made, or by combinations thereof. Geneexpression can be measured at the RNA level or the protein level by anymethod known in the art, including, without limitation, Northern blot,RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activityassay(s).

As used herein, the terms “homology-based gene silencing” or “HBGS” aregeneric tams that include both transcriptional gene silencing andpost-transcriptional gene silencing. Silencing of a target locus by anunlinked silencing locus can result from transcription inhibition(transcriptional gene silencing; TGS) or mRNA degradation(post-transcriptional gene silencing; PTGS), owing to the production ofdouble-stranded RNA (dsRNA) corresponding to promoter or transcribedsequences, respectively. Involvement of distinct cellular components ineach process suggests that dsRNA-induced TGS and PTGS likely result fromthe diversification of an ancient common mechanism. However, a strictcomparison of TGS and PTGS has been difficult to achieve because itgenerally relies on the analysis of distinct silencing loci. A singletransgene locus can be described to trigger both TGS and PTGS, owing tothe production of dsRNA corresponding to promoter and transcribedsequences of different target genes.

As used herein, the terms “nucleic acid molecule,” “nucleic acid,” or“polynucleotide” (all three terms are synonymous with one another) referto a polymeric form of nucleotides, which may include both sense andanti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms, andmixed polymers thereof. “A nucleotide” may refer to a ribonucleotide,deoxyribonucleotide, or a modified form of either type of nucleotide. Anucleic acid molecule is usually at least 10 bases in length, unlessotherwise specified. The terms may refer to a molecule of RNA or DNA ofindeterminate length. The terms include single- and double-strandedforms of DNA. A nucleic acid molecule may include either or bothnaturally-occurring and modified nucleotides linked together bynaturally occurring and/or non-naturally occurring nucleotide linkages.

Nucleic acid molecules may be modified chemically or biochemically, ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those of skill in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications (e.g., uncharged linkages: for example, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.;charged linkages: for example, phosphorothioates, phosphorodithioates,etc.; pendent moieties: for example, peptides; intercalators: forexample, acridine, psoralen, etc.; chelators; alkylators; and modifiedlinkages: for example, alpha anomeric nucleic acids, etc.). The term“nucleic acid molecule” also includes any topological conformation,including single-stranded, double-stranded, partially duplexed,triplexed, hairpinned, circular, and padlocked conformations.

Transcription proceeds in a 5′ to 3′ manner along a DNA strand. Thisindicates that RNA is made by sequential addition ofribonucleotide-5′-triphosphates to the 3′ terminus of the growing chain(with a requisite elimination of the pyrophosphate). In either a linearor circular nucleic acid molecule, discrete elements (e.g., particularnucleotide sequences) may be referred to as being “upstream” relative toa further element if they are bonded or would be bonded to the samenucleic acid in the 5′ direction from that element. Similarly, discreteelements may be “downstream” relative to a further element if they areor would be bonded to the same nucleic acid in the 3′ direction fromthat element.

As used herein, the term “base position” refers to the location of agiven base or nucleotide residue within a designated nucleic acid. Adesignated nucleic acid may be defined by alignment with a referencenucleic acid.

As used herein, the term “hybridization” refers to a process whereoligonucleotides and their analogs hybridize by hydrogen bonding, whichincludes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary bases. Generally, nucleic acid molecules consistof nitrogenous bases that are either pyrimidines (cytosine (C), uracil(U), and thymine (T)) or purines (adenine (A) and guanine (G)). Thesenitrogenous bases form hydrogen bonds between a pyrimidine and a purine,and bonding of a pyrimidine to a purine is referred to as “basepairing.” More specifically, A will hydrogen bond to T or U, and G willbond to C. “Complementary” refers to the base pairing that occursbetween two distinct nucleic acid sequences or two distinct regions ofthe same nucleic acid sequence.

As used herein, the terms “specifically hybridizable” and “specificallycomplementary” refers to a sufficient degree of complementarity suchthat stable and specific binding occurs between an oligonucleotide andthe DNA or RNA target. Oligonucleotides need not be 100% complementaryto the target sequence to specifically hybridize. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget DNA or RNA molecule interferes with the normal function of thetarget DNA or RNA, and there is sufficient degree of complementarity toavoid non-specific binding of an oligonucleotide to non-target sequencesunder conditions where specific binding is desired, for example, underphysiological conditions in the case of in vivo assays or systems. Suchbinding is referred to as specific hybridization. Hybridizationconditions resulting in particular degrees of stringency will varydepending upon the nature of the chosen hybridization method and thecomposition and length of the hybridizing nucleic acid sequences.Generally, the temperature of hybridization and the ionic strength(especially Na′ and/or Mgt′ concentration) of a hybridization bufferwill contribute to the stringency of hybridization, though wash timesalso influence stringency. Calculations regarding hybridizationconditions required for attaining particular degrees of stringency arediscussed in Sambrook et al. (ed.), Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

As used herein, the term “stringent conditions” encompasses conditionsunder which hybridization will only occur if there is less than 50%mismatch between the hybridization molecule and the DNA target.“Stringent conditions” include further particular levels of stringency.Thus, as used herein, “moderate stringency” conditions are those underwhich molecules with more than 50% sequence mismatch will not hybridize;conditions of “high stringency” are those under which sequences withmore than 20% mismatch will not hybridize; and conditions of “very highstringency” are those under which sequences with more than 10% mismatchwill not hybridize.

In particular embodiments, stringent conditions can includehybridization at 65° C., followed by washes at 65° C. with 0.1×SSC/0.1%SDS for 40 minutes. The following are representative, non-limitinghybridization conditions:

Very High Stringency: hybridization in 5×SSC buffer at 65° C. for 16hours; wash twice in 2×SSC buffer at room temperature for 15 minuteseach; and wash twice in 0.5×SSC buffer at 65° C. for 20 minutes each.

High Stringency: Hybridization in 5-6×SSC buffer at 65-70° C. for 16-20hours; wash twice in 2×SSC buffer at room temperature for 5-20 minuteseach; and wash twice in 1×SSC buffer at 55-70° C. for 30 minutes each.

Moderate Stringency: Hybridization in 6×SSC buffer at room temperatureto 55° C. for 16-20 hours; wash at least twice in 2-3×SSC buffer at roomtemperature to 55° C. for 20-30 minutes each.

In an embodiment, specifically hybridizable nucleic acid molecules canremain bound under very high stringency hybridization conditions. In anembodiment, specifically hybridizable nucleic acid molecules can remainbound under high stringency hybridization conditions. In an embodiment,specifically hybridizable nucleic acid molecules can remain bound undermoderate stringency hybridization conditions.

As used herein, the term “oligonucleotide” refers to a short nucleicacid polymer. Oligonucleotides may be formed by cleavage of longernucleic acid segments, or by polymerizing individual nucleotideprecursors. Automated synthesizers allow the synthesis ofoligonucleotides up to several hundred base pairs in length. Becauseoligonucleotides may bind to a complementary nucleotide sequence, theymay be used as probes for detecting DNA or RNA. Oligonucleotidescomposed of DNA (oligodeoxyribonucleotides) may be used in polymerasechain reaction, a technique for the amplification of small DNAsequences. In polymerase chain reaction, an oligonucleotide is typicallyreferred to as a “primer” which allows a DNA polymerase to extend theoligonucleotide and replicate the complementary strand.

As used herein, the terms “Polymerase Chain Reaction” or “PCR” refer toa procedure or technique in which minute amounts of nucleic acid, RNAand/or DNA, are amplified as described in U.S. Pat. No. 4,683,195.Generally, sequence information from the ends of the region of interestor beyond needs to be available, such that oligonucleotide primers canbe designed; these primers will be identical or similar in sequence toopposite strands of the template to be amplified. The 5′ terminalnucleotides of the two primers may coincide with the ends of theamplified material. PCR can be used to amplify specific RNA sequences,specific DNA sequences from total genomic DNA, and cDNA transcribed fromtotal cellular RNA, bacteriophage or plasmid sequences, etc. Seegenerally Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263(1987); Erlich, ed., PCR Technology, (Stockton Press, N Y, 1989).

As used herein, the term “primer” refers to an oligonucleotide capableof acting as a point of initiation of synthesis along a complementarystrand when conditions are suitable for synthesis of a primer extensionproduct. The synthesizing conditions include the presence of fourdifferent deoxyribonucleotide triphosphates and at least onepolymerization-inducing agent such as reverse transcriptase or DNApolymerase. These are present in a suitable buffer that may includeconstituents which are co-factors or which affect conditions such as pHand the like at various suitable temperatures. A primer is preferably asingle strand sequence, such that amplification efficiency is optimized,but double stranded sequences can be utilized.

As used herein, the term “probe” refers to an oligonucleotide orpolynucleotide sequence that hybridizes to a target sequence. In theTAQMAN® or TAQMAN®-style assay procedure, the probe hybridizes to aportion of the target situated between the annealing site of the twoprimers. A probe includes about eight nucleotides, about tennucleotides, about fifteen nucleotides, about twenty nucleotides, aboutthirty nucleotides, about forty nucleotides, or about fifty nucleotides.In some embodiments, a probe includes from about eight nucleotides toabout fifteen nucleotides.

In the Southern blot assay procedure, the probe hybridizes to a DNAfragment that is attached to a membrane. A probe includes about tennucleotides, about 100 nucleotides, about 250 nucleotides, about 500nucleotides, about 1,000 nucleotides, about 2,500 nucleotides, or about5,000 nucleotides. In some embodiments, a probe includes from about 500nucleotides to about 2,500 nucleotides.

A probe can further include a detectable label, e.g., a radioactivelabel, a biotinylated label, a fluorophore (TEXAS-RED®, fluoresceinisothiocyanate, etc.). The detectable label can be covalently attacheddirectly to the probe oligonucleotide, e.g., located at the probe's 5′end or at the probe's 3′ end. A probe including a fluorophore may alsofurther include a quencher, e.g., BLACK HOLE QUENCHER®, IOWA BLACK™,etc.

As used herein, the terms “sequence identity” or “identity” can be usedinterchangeably and refer to nucleic acid residues in two sequences thatshare similar base compositions when aligned for maximum correspondenceover a specified comparison window for either a polynucleotide orprotein fragment.

As used herein, the tam “percentage of sequence identity” refers to avalue determined by comparing two optimally aligned sequences (e.g.,nucleic acid sequences or amino acid sequences) over a comparisonwindow, wherein the portion of a sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to a referencesequence (that does not comprise additions or deletions) for optimalalignment of the two sequences. A percentage is calculated bydetermining the number of positions at which an identical nucleic acidor amino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the comparison window, and multiplying the resultby 100 to yield the percentage of sequence identity. Methods foraligning sequences for comparison are well known. Various programs andalignment algorithms are described in, for example: Smith and Waterman(1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol.48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444;Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS5:151-3; Corpet et al., (1988) Nucleic Acids Res. 16:10881-90; Huang etal., (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al., (1994) MethodsMol. Biol. 24:307-31; Tatiana et al., (1999) FEMS Microbiol. Lett.174:247-50.

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST; Altschul et al., (1990) J. Mol. Biol.215:403-10) is available from several sources, including the NationalCenter for Biotechnology Information (Bethesda, Md.), and on theinternet, for use in connection with several sequence analysis programs.A description of how to determine sequence identity using this programis available on the internet under the “help” section for BLAST. Forcomparisons of nucleic acid sequences, the “Blast 2 sequences” functionof the BLAST (Blastn) program may be employed using the defaultparameters. Nucleic acid sequences with even greater similarity to thereference sequences will show increasing percentage identity whenassessed by this method.

As used herein, the term “operably linked” refers to a nucleic acidplaced into a functional relationship with another nucleic acid.Generally, “operably linked” can mean that linked nucleic acids arecontiguous. Linking can be accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers are ligated or annealed to the nucleic acid and usedto link the contiguous polynucleotide fragment. However, elements neednot be contiguous to be operably linked.

As used herein, the term “promoter” refers to a region of DNA thatgenerally is located upstream (towards the 5′ region of a gene) of agene and is needed to initiate and drive transcription of the gene. Apromoter may permit proper activation or repression of a gene that itcontrols. A promoter may contain specific sequences that are recognizedby transcription factors. These factors may bind to a promoter DNAsequence, which results in the recruitment of RNA polymerase, an enzymethat synthesizes RNA from the coding region of the gene. The promotergenerally refers to all gene regulatory elements located upstream of thegene, including, upstream promoters, 5′-UTR, introns, and leadersequences.

As used herein, the term “upstream-promoter” refers to a contiguouspolynucleotide sequence that is sufficient to direct initiation oftranscription. As used herein, an upstream-promoter encompasses the siteof initiation of transcription with several sequence motifs, whichinclude TATA Box, initiator sequence, TFIIB recognition elements andother promoter motifs (Jennifer, E. F. et al., (2002) Genes & Dev., 16:2583-2592). The upstream promoter provides the site of action to RNApolymerase II which is a multi-subunit enzyme with the basal or generaltranscription factors like, TFIIA, B, D, E, F and H. These factorsassemble into a transcription pre initiation complex that catalyzes thesynthesis of RNA from DNA template.

The activation of the upstream-promoter is done by the additionalsequence of regulatory DNA sequence elements to which various proteinsbind and subsequently interact with the transcription initiation complexto activate gene expression. These gene regulatory elements sequencesinteract with specific DNA-binding factors. These sequence motifs maysometimes referred to as cis-elements. Such cis-elements, to whichtissue-specific or development-specific transcription factors bind,individually or in combination, may determine the spatiotemporalexpression pattern of a promoter at the transcriptional level. Thesecis-elements vary widely in the type of control they exert on operablylinked genes. Some elements act to increase the transcription ofoperably-linked genes in response to environmental responses (e.g.,temperature, moisture, and wounding). Other cis-elements may respond todevelopmental cues (e.g., germination, seed maturation, and flowering)or to spatial information (e.g., tissue specificity). See, for example,Langridge et al., (1989) Proc. Natl. Acad. Sci. USA 86:3219-23. Thesecis-elements are located at a varying distance from transcription startpoint, some cis-elements (called proximal elements) are adjacent to aminimal core promoter region while other elements can be positionedseveral kilobases upstream or downstream of the promoter (enhancers).

A “DNA binding transgene” is a polynucleotide coding sequence thatencodes a DNA binding protein. The DNA binding protein is subsequentlyable to bind to another molecule. A binding protein can bind to, forexample, a DNA molecule (a DNA-binding protein), an RNA molecule (anRNA-binding protein) and/or a protein molecule (a protein-bindingprotein). In the case of a protein-binding protein, it can bind toitself (to form homodimers, homotrimers, etc.) and/or it can bind to oneor more molecules of a different protein or proteins. A binding proteincan have more than one type of binding activity. For example, zincfinger proteins have DNA-binding, RNA-binding and protein-bindingactivity.

Examples of DNA binding proteins include; meganucleases, zinc fingers,CRISPRs and TALE binding domains that can be “engineered” to bind to apredetermined nucleotide sequence. Typically, the engineered DNA bindingproteins (e.g., zinc fingers, CRISPRs, or TALEs) are proteins that arenon-naturally occurring. Non-limiting examples of methods forengineering DNA-binding proteins are design and selection. A designedDNA binding protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing information in a database storinginformation of existing ZFP, CRISPR, and/or TALE designs and bindingdata. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536and WO 03/016496 and U.S. Publication Nos. 20110301073, 20110239315 and20119145940.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP. Zinc finger bindingdomains can be “engineered” to bind to a predetermined nucleotidesequence. Non-limiting examples of methods for engineering zinc fingerproteins are design and selection. A designed zinc finger protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPdesigns and binding data. See, for example, U.S. Pat. Nos. 6,140,081;6,453,242; 6,534,261 and 6,794,136; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496.

In other examples, the DNA-binding domain of one or more of thenucleases comprises a naturally occurring or engineered (non-naturallyoccurring) TAL effector DNA binding domain. See, e.g., U.S. PatentPublication No. 20110301073, incorporated by reference in its entiretyherein. The plant pathogenic bacteria of the genus Xanthomonas are knownto cause many diseases in important crop plants. Pathogenicity ofXanthomonas depends on a conserved type III secretion (T3S) system whichinjects more than different effector proteins into the plant cell. Amongthese injected proteins are transcription activator-like (TALEN)effectors which mimic plant transcriptional activators and manipulatethe plant transcriptome (see Kay et al., (2007) Science 318:648-651).These proteins contain a DNA binding domain and a transcriptionalactivation domain. One of the most well characterized TAL-effectors isAvrBs3 from Xanthomonas campestgris pv. Vesicatoria (see Bonas et al.,(1989) Mol Gen Genet 218: 127-136 and WO2010079430). TAL-effectorscontain a centralized domain of tandem repeats, each repeat containingapproximately 34 amino acids, which are key to the DNA bindingspecificity of these proteins. In addition, they contain a nuclearlocalization sequence and an acidic transcriptional activation domain(for a review see Schornack S, et al., (2006) J Plant Physiol 163(3):256-272). In addition, in the phytopathogenic bacteria Ralstoniasolanacearum two genes, designated brg11 and hpx17 have been found thatare homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar strain GMI1000 and in the biovar 4 strain RS1000(see Heuer et al., (2007) Appl and Enviro Micro 73(13): 4379-4384).These genes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas. See, e.g., U.S. Patent Publication No.20110301073, incorporated by reference in its entirety.

Specificity of these TAL effectors depends on the sequences found in thetandem repeats. The repeated sequence comprises approximately 102 bp andthe repeats are typically 91-100% homologous with each other (Bonas etal. ibid). Polymorphism of the repeats is usually located at positions12 and 13 and there appears to be a one-to-one correspondence betweenthe identity of the hypervariable diresidues at positions 12 and 13 withthe identity of the contiguous nucleotides in the TAL-effector's targetsequence (see Moscou and Bogdanove, (2009) Science 326:1501 and Boch etal., (2009) Science 326:1509-1512). Experimentally, the natural code forDNA recognition of these TAL-effectors has been determined such that anHD sequence at positions 12 and 13 leads to a binding to cytosine (C),NG binds to T, NI to A, C, G or T, NN binds to A or G, and ING binds toT. These DNA binding repeats have been assembled into proteins with newcombinations and numbers of repeats, to make artificial transcriptionfactors that are able to interact with new sequences and activate theexpression of a non-endogenous reporter gene in plant cells (Boch et al.ibid). Engineered TAL proteins have been linked to a FokI cleavage halfdomain to yield a TAL effector domain nuclease fusion (TALEN) exhibitingactivity in a yeast reporter assay (plasmid based target).

The CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR Associated) nuclease system is a recentlyengineered nuclease system based on a bacterial system that can be usedfor genome engineering. It is based on part of the adaptive immuneresponse of many bacteria and Archea. When a virus or plasmid invades abacterium, segments of the invader's DNA are converted into CRISPR RNAs(crRNA) by the ‘immune’ response. This crRNA then associates, through aregion of partial complementarity, with another type of RNA calledtracrRNA to guide the Cas9 nuclease to a region homologous to the crRNAin the target DNA called a “protospacer.” Cas9 cleaves the DNA togenerate blunt ends at the DSB at sites specified by a 20-nucleotideguide sequence contained within the crRNA transcript. Cas9 requires boththe crRNA and the tracrRNA for site specific DNA recognition andcleavage. This system has now been engineered such that the crRNA andtracrRNA can be combined into one molecule (the “single guide RNA”), andthe crRNA equivalent portion of the single guide RNA can be engineeredto guide the Cas9 nuclease to target any desired sequence (see Jinek etal., (2012) Science 337, p. 816-821, Jinek et al., (2013), eLife2:e00471, and David Segal, (2013) eLife 2:e00563). Thus, the CRISPR/Cassystem can be engineered to create a double-stranded break (DSB) at adesired target in a genome, and repair of the DSB can be influenced bythe use of repair inhibitors to cause an increase in error prone repair.

In other examples, the DNA binding transgene is a site specific nucleasethat comprises an engineered (non-naturally occurring) Meganuclease(also described as a homing endonuclease). The recognition sequences ofhoming endonucleases or meganucleases such as I-SceI, I-CeuI, PI-PspI,PI-Sce, I-SceIV, I-CsmI, I-PauI, I-PpoI, I-SceIII, I-CreI, I-TeeI,I-TevII and I-TevIII are known. See also U.S. Pat. No. 5,420,032; U.S.Pat. No. 6,833,252; Belfort et al., (1997) Nucleic Acids Res. 25:3379-303388; Dujon et al., (1989) Gene 82:115-118; Perler et al., (1994)Nucleic Acids Res. 22, 11127; Jasin (1996) Trends Genet. 12:224-228;Gimble et al., (1996) J. Mol. Biol. 263:163-180; Argast et al., (1998)J. Mol. Biol. 280:345-353 and the New England Biolabs catalogue. Inaddition, the DNA-binding specificity of horning endonucleases andmeganucleases can be engineered to bind non-natural target sites. See,for example, Chevalier et al., (2002) Molec. Cell 10:895-905; Epinat etal., (2003) Nucleic Acids Res. 5 31:2952-2962; Ashworth et al., (2006)Nature 441:656-659; Paques et al., (2007) Current Gene Therapy 7:49-66;U.S. Patent Publication No. 20070117128. The DNA-binding domains of thehoming endonucleases and meganucleases may be altered in the context ofthe nuclease as a whole (i.e., such that the nuclease includes thecognate cleavage domain) or may be fused to a heterologous cleavagedomain.

As used herein, the term “transformation” encompasses all techniquesthat a nucleic acid molecule can be introduced into such a cell.Examples include, but are not limited to: transfection with viralvectors; transformation with plasmid vectors; electroporation;lipofection; microinjection (Mueller et al., (1978) Cell 15:579-85);Agrobacterium-mediated transfer; direct DNA uptake; WHISKERS™-mediatedtransformation; and microprojectile bombardment. These techniques may beused for both stable transformation and transient transformation of aplant cell. “Stable transformation” refers to the introduction of anucleic acid fragment into a genome of a host organism resulting ingenetically stable inheritance. Once stably transformed, the nucleicacid fragment is stably integrated in the genome of the host organismand any subsequent generation. Host organisms containing the transformednucleic acid fragments are referred to as “transgenic” organisms.“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

As used herein, the term “transgene” refers to an exogenous nucleic acidsequence. In one example, a transgene is a gene sequence (e.g., anherbicide-resistance gene), a gene encoding an industrially orpharmaceutically useful compound, or a gene encoding a desirableagricultural trait. In yet another example, a transgene is a small RNA,such as an antisense nucleic acid sequence, wherein expression of thesmall RNA sequence inhibits expression of a target nucleic acidsequence. A transgene may contain regulatory sequences operably linkedto the transgene (e.g., a promoter, intron, or 3′-UTR). In someembodiments, a nucleic acid of interest (or alternatively described as agene of interest) is a transgene. However, in other embodiments, anucleic acid of interest is an endogenous nucleic acid, whereinadditional genomic copies of the endogenous nucleic acid are desired, ora nucleic acid that is in the antisense orientation with respect to thesequence of a target nucleic acid in a host organism.

As used herein, the term “small RNA” refers to several classes ofnon-coding ribonucleic acid (ncRNA). The term small RNA describes theshort chains of ncRNA produced in bacterial cells, animals, plants, andfungi. These short chains of ncRNA may be produced naturally within thecell or may be produced by the introduction of an exogenous sequencethat expresses the short chain or ncRNA. The small RNA sequences do notdirectly code for a protein, and differ in function from other RNA inthat small RNA sequences are only transcribed and not translated. Thesmall RNA sequences are involved in other cellular functions, includinggene expression and modification. Small RNA molecules are usually madeup of about 20 to 30 nucleotides. The small RNA sequences may be derivedfrom longer precursors. The precursors form structures that fold back oneach other in self-complementary regions; they are then processed by thenuclease Dicer in animals or DCL1 in plants.

Many types of small RNA exist either naturally or produced artificially,including microRNAs (miRNAs), short interfering RNAs (siRNAs), antisenseRNA, short hairpin RNA (shRNA), and small nucleolar RNAs (snoRNAs).Certain types of small RNA, such as microRNA, and siRNA, are importantin gene silencing and RNA interference (RNAi). Gene silencing is aprocess of genetic regulation in which a gene that would normally beexpressed is “turned off” by an intracellular element, in this case, thesmall RNA. The protein that would normally be formed by this geneticinformation is not formed due to interference, and the information codedin the gene is blocked from expression.

As used herein, the term “small RNA” encompasses RNA molecules describedin the literature as “tiny RNA” (Storz, (2002) Science 296:1260-3;Illangasekare et al., (1999) RNA 5:1482-1489); prokaryotic “small RNA”(sRNA) (Wassarman et al., (1999) Trends Microbiol. 7:37-45); eukaryotic“noncoding RNA (ncRNA)”; “micro-RNA (miRNA)”; “small non-mRNA (snmRNA)”;“functional RNA (fRNA)”; “transfer RNA (tRNA)”; “catalytic RNA” [e.g.,ribozymes, including self-acylating ribozymes (Illangaskare et al.,(1999) RNA 5:1482-1489); “small nucleolar RNAs (snoRNAs)”; “tmRNA”(a.k.a. “10S RNA,” Muto et al., (1998) Trends Biochem Sci. 23:25-29; andGillet et al., (2001) Mol Microbiol. 42:879-885); RNAi moleculesincluding without limitation “small interfering RNA (siRNA),”“endoribonuclease-prepared siRNA (e-siRNA),” “short hairpin RNA(shRNA),” and “small temporally regulated RNA (stRNA),” “diced siRNA(d-siRNA),” and aptamers, oligonucleotides and other synthetic nucleicacids that comprise at least one uracil base.

As used herein, the term “vector” refers to a nucleic acid molecule asintroduced into a cell, thereby producing a transformed cell. A vectormay include nucleic acid sequences that permit it to replicate in thehost cell, such as an origin of replication. Examples include, but arenot limited to, a plasmid, cosmid, bacteriophage, bacterial artificialchromosome (BAC), or virus that carries exogenous DNA into a cell. Avector can also include one or more genes, antisense molecules, and/orselectable marker genes and other genetic elements known in the art. Avector may transduce, transform, or infect a cell, thereby causing thecell to express the nucleic acid molecules and/or proteins encoded bythe vector. A vector may optionally include materials to aid inachieving entry of the nucleic acid molecule into the cell (e.g., aliposome).

As used herein, the terms “cassette,” “expression cassette,” and “geneexpression cassette” refer to a segment of DNA that can be inserted intoa nucleic acid or polynucleotide at specific restriction sites or byhomologous recombination. A segment of DNA comprises a polynucleotidecontaining a gene of interest that encodes a small RNA or a polypeptideof interest, and the cassette and restriction sites are designed toensure insertion of the cassette in the proper reading frame fortranscription and translation. In an embodiment, an expression cassettecan include a polynucleotide that encodes a small RNA or a polypeptideof interest and having elements in addition to the polynucleotide thatfacilitate transformation of a particular host cell. In an embodiment, agene expression cassette may also include elements that allow forenhanced expression of a small RNA or a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, an intron, a 5′ untranslated, a 3′ untranslated regionsequence, a terminator sequence, a polyadenylation sequence, and thelike.

As used herein, the term “heterologous coding sequence” is used toindicate any polynucleotide that codes for, or ultimately codes for, apeptide or protein or its equivalent amino acid sequence, e.g., anenzyme, that is not normally present in the host organism and can beexpressed in the host cell under proper conditions. As such,“heterologous coding sequences” may include one or additional copies ofcoding sequences that are not normally present in the host cell, suchthat the cell is expressing additional copies of a coding sequence thatare not normally present in the cells. The heterologous coding sequencescan be RNA or any type thereof, e.g., mRNA, DNA or any type thereof,e.g., cDNA, or a hybrid of RNA/DNA. Examples of coding sequencesinclude, but are not limited to, full-length transcription units thatcomprise such features as the coding sequence, introns, promoterregions, 5′-UTR, 30-UTRs and enhancer regions.

“Heterologous coding sequences” also includes the coding portion of thepeptide or enzyme, i.e., the cDNA or mRNA sequence, of the peptide orenzyme, as well as the coding portion of the full-length transcriptionalunit, i.e., the gene comprising introns and exons, as well as “codonoptimized” sequences, truncated sequences or other forms of alteredsequences that code for the enzyme or code for its equivalent amino acidsequence, provided that the equivalent amino acid sequence produces afunctional protein. Such equivalent amino acid sequences can have adeletion of one or more amino acids, with the deletion being N-terminal,C-terminal or internal. Truncated forms are envisioned as long as theyhave the catalytic capability indicated herein.

As used herein, the term “control” refers to a sample used in ananalytical procedure for comparison purposes. A control can be“positive” or “negative.” For example, where the purpose of ananalytical procedure is to detect a differentially expressed transcriptor polypeptide in cells or tissue, it is generally preferable to includea positive control, such as a sample from a known plant exhibiting thedesired expression, and a negative control, such as a sample from aknown plant lacking the desired expression.

As used herein, the term “plant” includes plants and plant partsincluding, but not limited to, plant cells and plant tissues such asleaves, stems, roots, flowers, pollen, and seeds. A class of plant thatcan be used in the present invention is generally as broad as the classof higher and lower plants amenable to mutagenesis includingangiosperms, gymnosperms, ferns and multicellular algae. Thus, “plant”includes dicot and monocot plants. Examples of dicotyledonous plantsinclude tobacco, Arabidopsis, soybean, tomato, papaya, canola,sunflower, cotton, alfalfa, potato, grapevine, pigeon pea, pea,Brassica, chickpea, sugar beet, rapeseed, watermelon, melon, pepper,peanut, pumpkin, radish, spinach, squash, broccoli, cabbage, carrot,cauliflower, celery, Chinese cabbage, cucumber, eggplant, and lettuce.Examples of monocotyledonous plants include corn, rice, wheat,sugarcane, barley, rye, sorghum, orchids, bamboo, banana, cattails,lilies, oat, onion, millet, and triticale.

As used herein, the term “plant material” refers to leaves, stems,roots, flowers or flower parts, fruits, pollen, egg cells, zygotes,seeds, cuttings, cell or tissue cultures, or any other part or productof a plant. In an embodiment, plant material includes cotyledon andleaf. In an embodiment, plant material includes seed, embryo, or ovule.In an embodiment, plant material includes root tissues and other planttissues located underground.

As used herein, the term “selectable marker gene” refers to a gene thatis optionally used in plant transformation to, for example, protectplant cells from a selective agent or provide resistance/tolerance to aselective agent. In addition, “selectable marker gene” is meant toencompass reporter genes. Only those cells or plants that receive afunctional selectable marker are capable of dividing or growing underconditions having a selective agent. Examples of selective agents caninclude, for example, antibiotics, including spectinomycin, neomycin,kanamycin, paromomycin, gentamicin, and hygromycin. These selectablemarkers include neomycin phosphotransferase (npt II), which expresses anenzyme conferring resistance to the antibiotic kanamycin, and genes forthe related antibiotics neomycin, paromomycin, gentamicin, and G418, orthe gene for hygromycin phosphotransferase (hpt), which expresses anenzyme conferring resistance to hygromycin. Other selectable markergenes can include genes encoding herbicide resistance including bar orpat (resistance against glufosinate ammonium or phosphinothricin),acetolactate synthase (ALS, resistance against inhibitors such assulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs),pyrimidinyl oxybenzoates (POBs), and sulfonylamino carbonyltriazolinones that prevent the first step in the synthesis of thebranched-chain amino acids), glyphosate, 2,4-D, and metal resistance orsensitivity. Examples of “reporter genes” that can be used as aselectable marker gene include the visual observation of expressedreporter gene proteins such as proteins encoding □-glucuronidase (GUS),luciferase, green fluorescent protein (GFP), yellow fluorescent protein(YFP), DsRed, □-galactosidase, chloramphenicol acetyltransferase (CAT),alkaline phosphatase, and the like. The phrase “marker-positive” refersto plants that have been transformed to include a selectable markergene.

As used herein, the term “detectable marker” refers to a label capableof detection, such as, for example, a radioisotope, fluorescentcompound, bioluminescent compound, a chemiluminescent compound, metalchelator, or enzyme. Examples of detectable markers include, but are notlimited to, the following: fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,□-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In anembodiment, a detectable marker can be attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

As used herein, the term “detecting” is used in the broadest sense toinclude both qualitative and quantitative measurements of a specificmolecule, for example, measurements of a specific polypeptide.

Unless otherwise specifically explained, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art that this disclosure belongs. Definitionsof common terms in molecular biology can be found in, for example:Lewin, Genes V, Oxford University Press, 1994; Kendrew et al. (eds.),The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994; andMeyers (ed.), Molecular Biology and Biotechnology: A Comprehensive DeskReference, VCH Publishers, Inc., 1995.

Regulatory Elements

Plant promoters used for basic research or biotechnological applicationsare generally unidirectional, directing the expression of a transgenethat has been fused at its 3′ end (downstream). It is often necessary torobustly express transgenes within plants for metabolic engineering andtrait stacking. In addition, multiple novel promoters are typicallyrequired in transgenic crops to drive the expression of multiple genes.Disclosed, herein is a promoter that can direct the expression of afirst gene that has been fused at its 3′ end (downstream).

Development of transgenic products is becoming increasingly complex,which requires robustly expressing transgenes and stacking multipletransgenes into a single locus. Traditionally, each transgene requires aunique promoter for expression wherein multiple promoters are requiredto express different transgenes within one gene stack. With anincreasing size of gene stacks, this frequently leads to repeated use ofthe same promoter to obtain similar levels of expression patterns ofdifferent transgenes for expression of a single polygenic trait.Multi-gene constructs driven by the same promoter are known to causegene silencing resulting in less efficacious transgenic products in thefield. Excess of transcription factor (TF)-binding sites due to promoterrepetition can cause depletion of endogenous TFs leading totranscriptional inactivation. The silencing of transgenes will likelyundesirably affect performance of a transgenic plant produced to expresstransgenes. Repetitive sequences within a transgene may lead to geneintra-locus homologous recombination resulting in polynucleotiderearrangements.

Tissue specific (i.e., tissue-preferred) or organ specific promotersdrive gene expression in a certain tissue such as in the ovule, embryo,seed, kernel, root, leaf or tapetum of the plant. Tissue anddevelopmental stage specific promoters derive the expression of genes,which are expressed in particular tissues or at particular time periodsduring plant development. Tissue specific promoters are required forcertain applications in the transgenic plants industry and are desirableas they permit specific expression of heterologous genes in a tissueand/or developmental stage selective manner, indicating expression ofthe heterologous gene differentially at a various organs, tissues and/ortimes, but not in other. For example, increased resistance of a plant toinfection by soil-borne pathogens might be accomplished by transformingthe plant genome with a pathogen-resistance gene such thatpathogen-resistance protein is robustly expressed within the roots ofthe plant. Alternatively, it may be desirable to express a transgene inplant tissues that are in a particular growth or developmental phasesuch as, for example, cell division or elongation. Another applicationis the desirability of using tissue specific promoters, e.g., such thatwould confine the expression of the transgenes encoding an agronomictrait in developing xylem. Another application is for driving expressionof a transgene within a seed, ovule, or embryo. One particular problemremaining in the identification of tissue specific promoters is how toidentify the potentially most important genes and their correspondingpromoters, and to relate these to specific developmental properties ofthe cell. Another problem is to clone all relevant cis-actingtranscriptional control elements so that the cloned DNA fragment drivestranscription in the wanted specific expression pattern. A particularproblem is to identify tissue-specific promoters, related to specificcell types, developmental stages and/or functions in the plant that arenot expressed in other plant tissues.

Provided are methods and constructs using Brassica napus GALE genepromoter regulatory elements to express transgenes in plant. In anembodiment, a promoter can be a Brassica napus GALE gene promoter of:

(SEQ ID NO: 1) caacaaaaatgcactttttcgccaaaaatacatttttcttcaaaaaccgcaaaaatattttctgccaaacccgtaaaaatactatttttctgccgaaacgtaaaaaaaaatattttaattattttattaacaagtccacttggatgtagatgaaaatttaaaaaatgaaaagcaaacgaacatagttgcattcagatgattcatctggatgcatggacgaaatgaagaaacgaacaacacccatatagagcatctggataagacatctagatggatcattacaaaagaacagggcctaaacatgtgagatgtttgaagcaatcagtcaaaagtaaccaccaaatcgaattatgaaagcgttgattggatggacaagtttaacaaccattgtttgattggacaacgccgttatctaaacttttagtgtgctgtgtacatcattactatgaatcagttagttaaaaatattatggtcagtgaatgacagtaagattacttcagaacttgagagatttaccgcaaaaagaaacacaataacgcgtaggaaaaatatcctctgttttttgcaattattctcgtagatttggttatcagtaggtatcacgttttacaaaaatagaattacaatacatgccgcaagaaaaagactttctctttttaatttccccaatttggttatcagtattcagtaagtttcacatttttacaaaaatataaattaaaatacatactgcaagaaaaatacttttttaatttcgccaatttggttatcagtagttttcacatttttacaaaaatataattaaaatataaactgcaacaaaaagacttatctttttaatttccccaatttggttatcagtattcagtaggtttcacatttacaaaaatattattaaaatacatactgcaagaaacatacctttttaatttcgccaatctggttatcagtagttttcacatttttacaaaaatagaattaaaatacaaactgcaacaaaaagacttatctttttaatttccaccaataagttatttatttatttaatcctcccgtgaggaaaaagacaagattgaggatgaatatacgtaactgaaaattgaggaaacagagccatcaacctttcaacacggatgatcatcatcatcactctctgccgcctttaaatagaaaccaacaaagacattcttgagcccacactcactcctttcctatttcttcgctttgcgtgccttccttccttcttatctacttgtatcccacaaaaagctacttaataccatttaataaagaccccaactttcttgtgtcttctctcttatcatcttcgctgtgatctctctgtctccctctctcttatccaaaagattagtataaaaggatcgatctttccttgtgggttcttccataaaacttcgattctcgact.

In an embodiment, a gene expression cassette comprises a promoter. In anembodiment, a promoter can be a Brassica napus GALE1 gene promoter ofthe subject disclosure. In an embodiment, a gene expression cassettecomprises a promoter, wherein the promoter is at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100%identical to SEQ ID NO:1. In an embodiment, a gene expression cassettecomprises a Brassica napus GALE1 gene promoter that is operably linkedto a transgene. In an embodiment, a gene expression cassette comprisingthe Brassica napus GALE1 gene promoter may drive expression of two ormore transgenes. In an illustrative embodiment, a gene expressioncassette comprises a Brassica napus GALE1 gene promoter that is operablylinked to a transgene, wherein the transgene can be an insecticidalresistance transgene, a herbicide tolerance transgene, a nitrogen useefficiency transgene, a water use efficiency transgene, a nutritionalquality transgene, a DNA binding transgene, a selectable markertransgene, or combinations thereof.

Transgene expression may also be regulated by a 3′-untranslated generegion (i.e., 3′-UTR) located downstream of the gene's coding sequence.Both a promoter and a 3′-UTR can regulate transgene expression. While apromoter is necessary to drive transcription, a 3′-UTR gene region canterminate transcription and initiate polyadenylation of a resulting mRNAtranscript for translation and protein synthesis. A 3′-UTR gene regionaids stable expression of a transgene. In an embodiment, a 3′-UTR can bea Brassica napus GALE1 gene 3′-UTR of:

(SEQ ID NO: 2) actttactctttctctctaatcgctcaatatacaaaagaaaagtgtttacatacacacatcatatatagtttgcttttagtttccatgtaaccgaacgggtctgtttacttctatgaataaaatagctagttgatgattctgttgattgatacactctatggatagttcaagattttattacaatccaacgatgatttgtatcaaatagagcccaccagatcaagaaagcatactccagaagcttttgttcaatctaccatcagataacatatcaataaccatcttcatggtggaaccatctgcagcaaacccacacctcttcatttcttctatgagttcaactgaagcgactacaccactacctccgagatgaactcggatcagtgtgttgtatgtacactcatttggcgcaatcccatcctcctctcccatctttttaaacaacatatccgcttcagacagtgagcctttcttacacagtcctgcaatcattatggt a.

In an embodiment, a gene expression cassette comprises a 3′-UTR. In anembodiment, a 3′-UTR can be a Brassica napus GALE1 gene 3′-UTR. In anembodiment, a gene expression cassette comprises a 3′-UTR, wherein the3′-UTR is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:2. In anembodiment, a gene expression cassette comprises a Brassica napus GALE1gene 3′-UTR that is operably linked to a transgene. In an illustrativeembodiment, a gene expression cassette comprises a 3′-UTR that isoperably linked to a transgene, wherein the transgene can be aninsecticidal resistance transgene, an herbicide tolerance transgene, anitrogen use efficiency transgene, a water use efficiency transgene, anutritional quality transgene, a DNA binding transgene, a selectablemarker transgene, or combinations thereof.

In an embodiment, a gene expression cassette comprises a promoter and a3′-UTR purified from the Brassica napus GALE1 gene. In an embodiment, agene expression cassette comprises: a) a promoter, wherein the promoteris at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, 99.8%, or 100% identical to SEQ ID NO:1, and/or, b) a 3′-UTR,wherein the 3′-UTR is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:2.

For example, a gene expression cassette may include both a promoter anda 3′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:1, andthe 3′-UTR is a polynucleotide of SEQ ID NO:2. A promoter and 3′-UTR canbe operably linked to different transgenes within a gene expressioncassette when a gene expression cassette includes one or moretransgenes. In an illustrative embodiment, a gene expression cassettecomprises a Brassica napus GALE1 gene promoter (SEQ ID NO:1) that isoperably linked to a transgene, wherein the transgene can be aninsecticidal resistance transgene, an herbicide tolerance transgene, anitrogen use efficiency transgene, a water use efficiency transgene, anutritional quality transgene, a DNA binding transgene, a selectablemarker transgene, or combinations thereof. In an illustrativeembodiment, a gene expression cassette comprises a Brassica napus GALE1gene 3′-UTR (SEQ ID NO:2) that is operably linked to a transgene,wherein the transgene can be an insecticidal resistance transgene, anherbicide tolerance transgene, a nitrogen use efficiency transgene, awater us efficiency transgene, a nutritional quality transgene, a DNAbinding transgene, a selectable marker transgene, or combinationsthereof.

In an embodiment, a vector comprises a gene expression cassette, asdisclosed herein. In an embodiment, a vector can be a plasmid, a cosmid,a bacterial artificial chromosome (BAC), a bacteriophage, a virus, or anexcised polynucleotide fragment for use in transformation or genetargeting such as a donor DNA.

In an embodiment, a cell or plant comprises a gene expression cassette,as disclosed herein. In an embodiment, a cell or plant comprises avector comprising a gene expression cassette, as disclosed herein. In anembodiment, a vector can be a plasmid, a cosmid, a bacterial artificialchromosome (BAC), a bacteriophage, or a virus. Thereby, a cell or plantcomprising a gene expression cassette, as disclosed herein, is atransgenic cell or transgenic plant, respectively. In an embodiment, atransgenic plant can be a monocotyledonous plant. In an embodiment, atransgenic monocotyledonous plant can be, but is not limited to, maize,wheat, rice, sorghum, oats, rye, bananas, sugar cane, and millet. In anembodiment, a transgenic plant can be a dicotyledonous plant. In anembodiment, a transgenic dicotyledonous plant can be, but is not limitedto, soybean, cotton, sunflower, and canola. An embodiment also includesa transgenic seed from a transgenic plant, as disclosed herein.

In an embodiment, a gene expression cassette includes two or moretransgenes. The two or more transgenes may be operably linked to aBrassica napus GALE1 gene promoter or 3′-UTR, as disclosed herein. In anembodiment, a gene expression cassette includes one or more transgenes.In an embodiment with one or more transgenes, at least one transgene isoperably linked to a Brassica napus GALE1 gene promoter or 3′-UTR or thesubject disclosure.

Selectable Markers

Various selectable markers, also described as reporter genes, can beincorporated into a chosen expression vector to allow for identificationand selection of transformed plants (“transformants”). Many methods areavailable to confirm expression of selectable markers in transformedplants, including, for example, DNA sequencing and PCR (polymerase chainreaction), Southern blotting, RNA blotting, immunological methods fordetection of a protein expressed from the vector, eg., precipitatedprotein that mediates phosphinothricin resistance, or visual observationof other proteins such as reporter genes encoding □-glucuronidase (GUS),luciferase, green fluorescent protein (GFP), yellow fluorescent protein(YFP), red fluorescent protein (DsRed), □-galactosidase, chloramphenicolacetyltransferase (CAT), alkaline phosphatase, and the like (seeSambrook, et al., Molecular Cloning: A Laboratory Manual, Third Edition,Cold Spring Harbor Press, N.Y., 2001, the content is incorporated hereinby reference in its entirety).

Selectable marker genes are utilized for selection of transformed cellsor tissues. Selectable marker genes include genes encoding antibioticresistance, such as those encoding neomycin phosphotransferase II(NptII) and hygromycin phosphotransferase (HPT) as well as genesconferring resistance to herbicidal compounds. Herbicide resistancegenes generally code for a modified target protein insensitive to theherbicide or for an enzyme that degrades or detoxifies the herbicide inthe plant before it can act. For example, resistance to glyphosate hasbeen obtained by using genes coding for mutant target enzymes,5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Genes and mutantsfor EPSPS are well known, and further described below. Resistance toglufosinate ammonium, bromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D)have been obtained by using bacterial genes encoding pat or DSM-2, anitrilase, an aad-1 or an aad-12 gene, which detoxifies the respectiveherbicides.

In an embodiment, herbicides can inhibit the growing point or meristem,including imidazolinone or sulfonylurea, and genes forresistance/tolerance of acetohydroxyacid synthase (AHAS) andacetolactate synthase (ALS) for these herbicides are well known.Glyphosate resistance genes include mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) and dgt-28 genes(via the introduction of recombinant nucleic acids and/or various formsof in vivo mutagenesis of native EPSPs genes), aroA genes and glyphosateacetyl transferase (GAT) genes, respectively). Resistance genes forother phosphono compounds include bar genes from Streptomyces species,including Streptomyces hygroscopicus and Streptomyces viridichromogenes,and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). Exemplary genes conferring resistance tocyclohexanediones and/or aryloxyphenoxypropanoic acid (includingHaloxyfop, Diclofop, Fenoxyprop, Fluazifop, Quizalofop) include genes ofacetyl coenzyme A carboxylase (ACCase)-Accl-S1, Accl-S2 and Accl-S3. Inan embodiment, herbicides can inhibit photosynthesis, including triazine(psbA and ls+ genes) or benzonitrile (nitrilase gene).

In an embodiment, selectable marker genes include, but are not limitedto, genes encoding: neomycin phosphotransferase II; cyanamide hydratase;aspartate kinase; dihydrodipicolinate synthase; tryptophandecarboxylase; dihydrodipicolinate synthase and desensitized aspartatekinase; bar gene; tryptophan decarboxylase; neomycin phosphotransferase(NEO); hygromycin phosphotransferase (HPT or HYG); dihydrofolatereductase (DHFR); phosphinothricin acetyltransferase;2,2-dichloropropionic acid dehalogenase; acetohydroxyacid synthase;5-enolpyruvyl-shikimate-phosphate synthase (aroA); haloarylnitrilase;acetyl-coenzyme A carboxylase; dihydropteroate synthase (sul I); and 32kD photosystem II polypeptide (psbA).

An embodiment also includes genes encoding resistance to: 2,4-D;chloramphenicol; methotrexate; hygromycin; spectinomycin; bromoxynil;glyphosate; and phosphinothricin.

The above list of selectable marker genes is not meant to be limiting.Any reporter or selectable marker gene are encompassed by the presentinvention.

Selectable marker genes are synthesized for optimal expression in aplant. For example, in an embodiment, a coding sequence of a gene hasbeen modified by codon optimization to enhance expression in plants. Aselectable marker gene can be optimized for expression in a particularplant species or alternatively can be modified for optimal expression indicotyledonous or monocotyledonous plants. Plant preferred codons may bedetermined from the codons of highest frequency in the proteinsexpressed in the largest amount in the particular plant species ofinterest. In an embodiment, a selectable marker gene is designed to beexpressed in plants at a higher level resulting in higher transformationefficiency. Methods for plant optimization of genes are well known.Guidance regarding the optimization and manufacture of syntheticpolynucleotide sequences can be found in, for example, WO2013016546,WO2011146524, WO1997013402, U.S. Pat. No. 6,166,302, and U.S. Pat. No.5,380,831, herein incorporated by reference.

Transgenes

The disclosed methods and compositions can be used to expresspolynucleotide gene sequences within the plant genome. Accordingly,expression of genes encoding herbicide tolerance, insect resistance,nutrients, antibiotics or therapeutic molecules can be driven by a plantpromoter.

In one embodiment the Brassica napus GALE1 gene regulatory element ofthe subject disclosure is combined or operably linked with gene encodingpolynucleotide sequences that provide resistance or tolerance toglyphosate or another herbicide, and/or provides resistance to selectinsects or diseases and/or nutritional enhancements, and/or improvedagronomic characteristics, and/or proteins or other products useful infeed, food, industrial, pharmaceutical or other uses. The transgenes canbe “stacked” with two or more nucleic acid sequences of interest withina plant genome. Stacking can be accomplished, for example, viaconventional plant breeding using two or more events, transformation ofa plant with a construct which contains the sequences of interest,re-transformation of a transgenic plant, or addition of new traitsthrough targeted integration via homologous recombination.

Such polynucleotide sequences of interest include, but are not limitedto, those examples provided below:

1. Genes or Coding Sequence (e.g., iRNA) That Confer Resistance to Pestsor Disease

(A) Plant Disease Resistance Genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. Examples of such genes include, the tomato Cf-9 genefor resistance to Cladosporium flavum (Jones et al., 1994 Science266:789), tomato Pto gene, which encodes a protein kinase, forresistance to Pseudomonas syringaepv. tomato (Martin et al., 1993Science 262:1432), and Arabidopsis RSSP2 gene for resistance toPseudomonas syringae (Mindrinos et al., 1994 Cell 78:1089).

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon, such as, a nucleotide sequence ofa Bt □-endotoxin gene (Geiser et al., 1986 Gene 48:109), and avegetative insecticidal (VIP) gene (see, e.g., Estruch et al., (1996)Proc. Natl. Acad. Sci. 93:5389-94). Moreover, DNA molecules encoding□-endotoxin genes can be purchased from American Type Culture Collection(Rockville, Md.), under ATCC accession numbers 40098, 67136, 31995 and31998.

(C) A lectin, such as, nucleotide sequences of several Clivia miniatamannose-binding lectin genes (Van Damme et al., 1994 Plant Molec. Biol.24:825).

(D) A vitamin binding protein, such as avidin and avidin homologs whichare useful as larvicides against insect pests. See U.S. Pat. No.5,659,026.

(E) An enzyme inhibitor, e.g., a protease inhibitor or an amylaseinhibitor. Examples of such genes include a rice cysteine proteinaseinhibitor (Abe et al., 19871 Biol. Chem. 262:16793), a tobaccoproteinase inhibitor I (Huub et al., 1993 Plant Molec. Biol. 21:985),and an □-amylase inhibitor (Sumitani et al., 1993 Biosci. Biotech.Biochem. 57:1243).

(F) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof, such as baculovirus expression of clonedjuvenile hormone esterase, an inactivator of juvenile hormone (Hammocket al., 1990 Nature 344:458).

(G) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest (J. Biol. Chem. 269:9).Examples of such genes include an insect diuretic hormone receptor(Regan, 1994), an allostatin identified in Diploptera punctata (Pratt,1989), and insect-specific, paralytic neurotoxins (U.S. Pat. No.5,266,361).

(H) An insect-specific venom produced in nature by a snake, a wasp,etc., such as a scorpion insectotoxic peptide (Pang, 1992 Gene 116:165).

(I) An enzyme responsible for a hyperaccumulation of monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(J) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, anuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. Examples ofsuch genes include, a callas gene (PCT published applicationWO93/02197), chitinase-encoding sequences (which can be obtained, forexample, from the ATCC under accession numbers 3999637 and 67152),tobacco hookworm chitinase (Kramer et al., 1993 Insect Molec. Biol.23:691), and parsley ubi4-2 polyubiquitin gene (Kawalleck et al., 1993Plant Molec. Biol. 21:673).

(K) A molecule that stimulates signal transduction. Examples of suchmolecules include nucleotide sequences for mung bean calmodulin cDNAclones (Botella et al., 1994 Plant Molec. Biol. 24:757) and a nucleotidesequence of a maize calmodulin cDNA clone (Griess et al., 1994 PlantPhysiol. 104:1467).

(L) A hydrophobic moment peptide. See U.S. Pat. Nos. 5,659,026 and5,607,914; the latter teaches synthetic antimicrobial peptides thatconfer disease resistance.

(M) A membrane permease, a channel former or a channel blocker, such asa cecropin-□ lytic peptide analog (Jaynes et al., 1993 Plant Sci. 89:43)which renders transgenic tobacco plants resistant to Pseudomonassolanacearum.

(N) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. See,for example, Beachy et al., (1990) Ann. Rev. Phytopathol. 28:451.

(O) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Forexample, Taylor et al., (1994) Abstract #497, Seventh Intl. Symposium onMolecular Plant-Microbe Interactions shows enzymatic inactivation intransgenic tobacco via production of single-chain antibody fragments.

(P) A virus-specific antibody. See, for example, Tavladoraki et al.,(1993) Nature 266:469, which shows that transgenic plants expressingrecombinant antibody genes are protected from virus attack

(Q) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo □-1,4-D polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-□-1,4-D-galacturonase (Lamb et al., 1992) Bio/Technology10:1436. The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart et al.,(1992 Plant J. 2:367).

(R) A developmental-arrestive protein produced in nature by a plant,such as the barley ribosome-inactivating gene that provides an increasedresistance to fungal disease (Longemann et al., 1992). Bio/Technology10:3305.

(S) RNA interference, in which an RNA molecule is used to inhibitexpression of a target gene. An RNA molecule in one example is partiallyor fully double stranded, which triggers a silencing response, resultingin cleavage of dsRNA into small interfering RNAs, which are thenincorporated into a targeting complex that destroys homologous mRNAs.See, e.g., Fire et al. U.S. Pat. No. 6,506,559; Graham et al. 6,573,099.

2. Genes that Confer Resistance to a Herbicide

(A) Genes encoding resistance or tolerance to a herbicide that inhibitsthe growing point or meristem, such as an imidazalinone, sulfonanilideor sulfonylurea herbicide. Exemplary genes in this category code formutant acetolactate synthase (ALS) (Lee et al., 1988 EMBOJ. 7:1241) alsoknown as acetohydroxyacid synthase (AHAS) enzyme (Mild et al., 1990Theor. Appl. Genet. 80:449).

(B) One or more additional genes encoding resistance or tolerance toglyphosate imparted by mutant EPSP synthase and aroA genes, or throughmetabolic inactivation by genes such as DGT-28, 2mEPSPS, GAT (glyphosateacetyltransferase) or GOX (glyphosate oxidase) and other phosphonocompounds such as glufosinate (pat, bar, and dsm-2 genes), andaryloxyphenoxypropionic acids and cyclohexanediones (ACCase inhibitorencoding genes). See, for example, U.S. Pat. No. 4,940,835, whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC Accession Number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061. Europeanpatent application No. 0 333 033 and U.S. Pat. No. 4,975,374 disclosenucleotide sequences of glutamine synthetase genes which conferresistance to herbicides such as L-phosphinothricin. The nucleotidesequence of a phosphinothricinacetyl-transferase gene is provided inEuropean application No. 0 242 246. De Greef et al., (1989)Bio/Technology 7:61 describes the production of transgenic plants thatexpress chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance toaryloxyphenoxypropionic acids and cyclohexanediones, such as sethoxydimand haloxyfop, are the Accl-S1, Accl-S2 and Accl-S3 genes described byMarshall et al., (1992) Theor. Appl. Genet. 83:435.

(C) Genes encoding resistance or tolerance to a herbicide that inhibitsphotosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibilla et al., (1991) Plant Cell 3:169describe the use of plasmids encoding mutant psbA genes to transformChlamydomonas. Nucleotide sequences for nitrilase genes are disclosed inU.S. Pat. No. 4,810,648, and DNA molecules containing these genes areavailable under ATCC accession numbers 53435, 67441 and 67442. Cloningand expression of DNA coding for a glutathione S-transferase isdescribed by Hayes et al., (1992) Biochem. J. 285:173.

(D) Genes encoding resistance or tolerance to a herbicide that bind tohydroxyphenylpyruvate dioxygenases (HPPD), enzymes which catalyze thereaction in which para-hydroxyphenylpyruvate (HPP) is transformed intohomogentisate. This includes herbicides such as isoxazoles (EP418175,EP470856, EP487352, EP527036, EP560482, EP682659, U.S. Pat. No.5,424,276), in particular isoxaflutole, which is a selective herbicidefor maize, diketonitriles (EP496630, EP496631), in particular2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-CF3 phenyl)propane-1,3-dione and2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-2,3Cl2phenyl) propane-1,3-dione,triketones (EP625505, EP625508, U.S. Pat. No. 5,506,195), in particularsulcotrione, and pyrazolinates. A gene that produces an overabundance ofHPPD in plants can provide tolerance or resistance to such herbicides,including, for example, genes described in U.S. Pat. Nos. 6,268,549 and6,245,968 and U.S. Patent Application, Publication No. 20030066102.

(E) Genes encoding resistance or tolerance to phenoxy auxin herbicides,such as 2,4-dichlorophenoxyacetic acid (2,4-D) and which may also conferresistance or tolerance to aryloxyphenoxypropionate (AOPP) herbicides.Examples of such genes include the □-ketoglutarate-dependent dioxygenaseenzyme (aad-1) gene, described in U.S. Pat. No. 7,838,733.

(F) Genes encoding resistance or tolerance to phenoxy auxin herbicides,such as 2,4-dichlorophenoxyacetic acid (2,4-D) and which may also conferresistance or tolerance to pyridyloxy auxin herbicides, such asfluroxypyr or triclopyr. Examples of such genes include the□-ketoglutarate-dependent dioxygenase enzyme gene (aad-12), described inWO 2007/053482 A2.

(G) Genes encoding resistance or tolerance to dicamba (see, e.g., U.S.Patent Publication No. 20030135879).

(H) Genes providing resistance or tolerance to herbicides that inhibitprotoporphyrinogen oxidase (PPO) (see U.S. Pat. No. 5,767,373).

(I) Genes providing resistance or tolerance to triazine herbicides (suchas atrazine) and urea derivatives (such as diuron) herbicides which bindto core proteins of photosystem II reaction centers (PS II) (seeBrussian et al., (1989) EMBO J. 1989, 8(4): 1237-1245.

3. Genes that Confer or Contribute to a Value-Added Trait

(A) Modified fatty acid metabolism, for example, by transforming maizeor Brassica with an antisense gene or stearoyl-ACP desaturase toincrease stearic acid content of the plant (Knultzon et al., 1992) Proc.Nat. Acad. Sci. USA 89:2624.

(B) Decreased phytate content

(1) Introduction of a phytase-encoding gene, such as the Aspergillusniger phytase gene (Van Hartingsveldt et al., 1993 Gene 127:87),enhances breakdown of phytate, adding more free phosphate to thetransformed plant.

(2) A gene could be introduced that reduces phytate content. In maize,this, for example, could be accomplished by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid (Raboy etal., 1990 Maydica 35:383).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. Examples of such enzymes include,Streptococcus mucus fructosyltransferase gene (Shiroza et al., 1988) J.Bacteriol. 170:810, Bacillus subtilis levansucrase gene (Steinmetz etal., 1985 Mol. Gen. Genel. 200:220), Bacillus licheniformis □-amylase(Pen et al., 1992 Bio/Technology 10:292), tomato invertase genes (Elliotet al., 1993), barley amylase gene (Sogaard et al., 1993 J. Biol. Chem.268:22480), and maize endosperm starch branching enzyme II (Fisher etal., 1993 Plant Physiol. 102:10450).

Transformation

Suitable methods for transformation of plants include any method thatDNA can be introduced into a cell, for example, and without limitation:electroporation (see, e.g., U.S. Pat. No. 5,384,253); micro-projectilebombardment (see, e.g., U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880;6,160,208; 6,399,861; and 6,403,865); Agrobacterium-mediatedtransformation (see, e.g., U.S. Pat. Nos. 5,635,055; 5,824,877;5,591,616; 5,981,840; and 6,384,301); and protoplast transformation(see, e.g., U.S. Pat. No. 5,508,184). These methods may be used tostably transform or transiently transform a plant.

A DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as agitation with silicon carbidefibers (see, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), or the DNAconstructs can be introduced directly to plant tissue using biolisticmethods, such as DNA particle bombardment (see, e.g., Klein et al.,(1987) Nature 327:70-73). Alternatively, the DNA construct can beintroduced into the plant cell via nanoparticle transformation (see,e.g., U.S. Patent Publication No. 2009/0104700, incorporated herein byreference in its entirety).

In addition, gene transfer may be achieved using non-Agrobacteriumbacteria or viruses such as Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, potato virus X, cauliflower mosaic virusand cassava vein mosaic virus and/or tobacco mosaic virus, see, e.g.,Chung et al., (2006) Trends Plant Sci. 11(1):1-4.

Through the application of transformation techniques, cells of virtuallyany plant species may be stably transformed, and these cells may bedeveloped into transgenic plants by well-known techniques. For example,techniques that may be particularly useful in the context of cottontransformation are described in U.S. Pat. Nos. 5,846,797; 5,159,135;5,004,863; and 6,624,344; techniques for transforming Brassica plants inparticular are described, for example, in U.S. Pat. No. 5,750,871;techniques for transforming soy bean are described, for example, in U.S.Pat. No. 6,384,301; and techniques for transforming maize are described,for example, in U.S. Pat. Nos. 7,060,876 and 5,591,616, andInternational PCT Publication WO 95/06722.

After effecting delivery of an exogenous nucleic acid to a recipientcell, a transformed cell is generally identified for further culturingand plant regeneration. In order to improve the ability to identifytransformants, one may desire to employ a selectable marker gene withthe transformation vector used to generate the transformant. In anillustrative embodiment, a transformed cell population can be assayed byexposing the cells to a selective agent or agents, or the cells can bescreened for the desired marker gene trait.

Cells that survive exposure to a selective agent, or cells that havebeen scored positive in a screening assay, may be cultured in media thatsupports regeneration of plants. In an embodiment, any suitable planttissue culture media may be modified by including further substances,such as growth regulators. Tissue may be maintained on a basic mediawith growth regulators until sufficient tissue is available to beginplant regeneration efforts, or following repeated rounds of manualselection, until the morphology of the tissue is suitable forregeneration (e.g., at least 2 weeks), then transferred to mediaconducive to shoot formation. Cultures are transferred periodicallyuntil sufficient shoot formation has occurred. Once shoots are formed,they are transferred to media conducive to root formation. Oncesufficient roots are formed, plants can be transferred to soil forfurther growth and maturity.

To confirm the presence of a desired nucleic acid comprising constructsprovided in regenerating plants, a variety of assays may be performed.Such assays may include: molecular biological assays, such as Southernand Northern blotting and PCR; biochemical assays, such as detecting thepresence of a protein product, e.g., by immunological methodology(ELISA, western blots, and/or LC-MS MS spectrophotometry) or byenzymatic function; plant part assays, such as leaf or root assays;and/or analysis of the phenotype of the whole regenerated plant.

Transgenic events may be screened, for example, by PCR amplificationusing, e.g., oligonucleotide primers specific for nucleic acid moleculesof interest. PCR genotyping is understood to include, but not be limitedto, polymerase-chain reaction (PCR) amplification of genomic DNA derivedfrom isolated host plant callus tissue predicted to contain a nucleicacid molecule of interest integrated into the genome, followed bystandard cloning and sequence analysis of PCR amplification products.Methods of PCR genotyping have been well described (see, e.g., Rios etal., (2002) Plant J. 32:243-53), and may be applied to genomic DNAderived from any plant species or tissue type, including cell cultures.Combinations of oligonucleotide primers that bind to both targetsequence and introduced sequence may be used sequentially or multiplexedin PCR amplification reactions. Oligonucleotide primers designed toanneal to the target site, introduced nucleic acid sequences, and/orcombinations of the two may be produced. Thus, PCR genotyping strategiesmay include, for example, and without limitation: amplification ofspecific sequences in the plant genome; amplification of multiplespecific sequences in the plant genome; amplification of non-specificsequences in the plant genome; and combinations of any of the foregoing.One skilled in the art may devise additional combinations of primers andamplification reactions to interrogate the genome. For example, a set offorward and reverse oligonucleotide primers may be designed to anneal tonucleic acid sequence(s) specific for the target outside the boundariesof the introduced nucleic acid sequence.

Forward and reverse oligonucleotide primers may be designed to annealspecifically to an introduced nucleic acid molecule, for example, at asequence corresponding to a coding region within a nucleotide sequenceof interest comprised therein, or other parts of the nucleic acidmolecule. Primers may be used in conjunction with primers describedherein. Oligonucleotide primers may be synthesized according to adesired sequence and are commercially available (e.g., from IntegratedDNA Technologies, Inc., Coralville, Iowa). Amplification may be followedby cloning and sequencing, or by direct sequence analysis ofamplification products. In an embodiment, oligonucleotide primersspecific for the gene target are employed in PCR amplifications.

Method of Expressing a Transgene

In an embodiment, a method of expressing at least one transgene in aplant comprises growing a plant comprising a Brassica napus GALE1 genepromoter operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene in a plant comprisinggrowing a plant comprising a Brassica napus GALE1 gene 3′-UTR operablylinked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene in a plant comprises growing a plantcomprising a Brassica napus GALE1 gene promoter and 3′-UTR operablylinked to at least one transgene.

In an embodiment, a method of expressing at least one transgene in aplant tissue or plant cell comprising culturing a plant tissue or plantcell comprising a Brassica napus GALE1 gene promoter operably linked toat least one transgene. In an embodiment, a method of expressing atleast one transgene in a plant tissue or plant cell comprising culturinga plant tissue or plant cell comprising a Brassica napus GALE1 gene3′-UTR operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene in a plant tissue or plantcell comprising culturing a plant tissue or plant cell comprising aBrassica napus GALE1 gene promoter and a Brassica napus GALE1 gene3′-UTR operably linked to at least one transgene.

In an embodiment, a method of expressing at least one transgene in aplant comprises growing a plant comprising a gene expression cassettecomprising a Brassica napus GALE1 gene promoter operably linked to atleast one transgene. In an embodiment, a method of expressing at leastone transgene in a plant comprises growing a plant comprising a geneexpression cassette comprising a Brassica napus GALE1 gene 3′-UTRoperably linked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene in a plant comprises growing a plantcomprising a gene expression cassette comprising a Brassica napus GALE1gene promoter and a Zea mays chlorophyll a/b binding gene 3′-UTRoperably linked to at least one transgene.

In an embodiment, a method of expressing at least one transgene in aplant tissue or plant cell comprises culturing a plant tissue or plantcell comprising a gene expression cassette a Brassica napus GALE1 genepromoter operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene in a plant tissue or plantcell comprises culturing a plant tissue or plant cell comprising a geneexpression cassette a Brassica napus GALE1 gene 3′-UTR operably linkedto at least one transgene. In an embodiment, a method of expressing atleast one transgene in a plant tissue or plant cell comprises culturinga plant tissue or plant cell comprising a gene expression cassette aBrassica napus GALE1 gene promoter and a Brassica napus GALE1 gene3′-UTR operably linked to at least one transgene.

In an embodiment, a plant, plant tissue, or plant cell comprises aBrassica napus GALE1 gene promoter (also including anupstream-promoter). In an embodiment, a Brassica napus GALE1 genepromoter can be SEQ ID NO:1. In an embodiment, a plant, plant tissue, orplant cell comprises a gene expression cassette comprising Brassicanapus GALE1 gene promoter, wherein the promoter is at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100%identical to SEQ ID NO:1. In an embodiment, a plant, plant tissue, orplant cell comprises a gene expression cassette comprising a Brassicanapus GALE1 gene promoter that is operably linked to a transgene. In anillustrative embodiment, a plant, plant tissue, or plant cell comprisesa gene expression cassette comprising a Brassica napus GALE1 genepromoter that is operably linked to a transgene, wherein the transgenecan be an insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, aselectable marker transgene, or combinations thereof.

In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a Brassica napus GALE1 gene 3′-UTR. In anembodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a Brassica napus GALE1 gene 3′-UTR. In anembodiment, the Brassica napus GALE1 gene 3′-UTR is a polynucleotide ofSEQ ID NO:2. In an embodiment, a plant, plant tissue, or plant cellcomprises a gene expression cassette comprising a Brassica napus GALE1gene 3′-UTR, wherein the Brassica napus GALE1 gene 3′-UTR is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identical to SEQ ID NO:2. In an embodiment, a geneexpression cassette comprises a Brassica napus GALE1 gene 3′-UTR that isoperably linked to a promoter, wherein the promoter is a Brassica napusGALE1 gene promoter, or a promoter that originates from a plant (e.g.,Arabidopsis thahana ubiquitin 10 promoter), a virus (e.g., Cassava veinmosaic virus promoter) or a bacteria (e.g., Agrobacterium tumefaciensdelta mas). In an embodiment, a plant, plant tissue, or plant cellcomprises a gene expression cassette comprising a Brassica napus GALE1gene 3′-UTR that is operably linked to a transgene. In an illustrativeembodiment, a plant, plant tissue, or plant cell comprising a geneexpression cassette comprising a Brassica napus GALE1 gene 3′-UTR thatis operably linked to a transgene, wherein the transgene can be aninsecticidal resistance transgene, an herbicide tolerance transgene, anitrogen use efficiency transgene, a water use efficiency transgene, anutritional quality transgene, a DNA binding transgene, a selectablemarker transgene, or combinations thereof.

In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a Brassica napus GALE1 gene promoter andBrassica napus GALE1 gene 3′-UTR that are operably linked to atransgene. The promoter and 3′-UTR can be operably linked to differenttransgenes within a gene expression cassette when a gene expressioncassette includes two or more transgenes. In an illustrative embodiment,a gene expression cassette comprises a Brassica napus GALE1 genepromoter that is operably linked to a transgene, wherein the transgenecan be an insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, aselectable marker transgene, or combinations thereof. In an illustrativeembodiment, a gene expression cassette comprises a Brassica napus GALE1gene 3′-UTR that is operably linked to a transgene, wherein thetransgene can be an insecticidal resistance transgene, an herbicidetolerance transgene, a nitrogen use efficiency transgene, a water useefficiency transgene, a nutritional quality transgene, a DNA bindingtransgene, a selectable marker transgene, or combinations thereof.

In an embodiment, transgene expression using methods described herein isexpressed within a plant's ovule and seed tissues. In an embodiment,transgene expression includes more than one transgene expressed in theplant's ovule and seed tissues. In an embodiment, a method of growing atransgenic plant as described herein includes ovule and seed-preferredtransgene expression. In an embodiment, a method of expressing atransgene in a plant tissue or plant cell includes ovule andseed-preferred tissues and ovule and seed-preferred cells. In anembodiment, the ovule and seed-preferred expression includesdicotyledonous leaf and stem-preferred expression.

In a further embodiment, transgene expression using methods describedherein is expressed within above ground plant tissues (e.g., ovule orseed). In an embodiment, transgene expression includes more than onetransgene expressed in above ground plant tissues such as ovule or seed.In other embodiments, the expression of the transgene is within theendosperm tissue of seeds. In an embodiment, a method of growing atransgenic plant as described herein includes above ground plant tissuestransgene expression. In an embodiment, a method of expressing atransgene in a plant tissue or plant cell above ground plant tissues andabove ground plant cells. In an embodiment, the above ground planttissue expression includes dicotyledonous above ground plant tissueexpression.

In an embodiment, a plant, plant tissue, or plant cell comprises avector comprising a Brassica napus GALE1 gene promoter, or 3′-UTRregulatory element, as disclosed herein. In an embodiment, a plant,plant tissue, or plant cell comprises a vector comprising a Brassicanapus GALE1 gene promoter, or 3′-UTR regulatory element, as disclosedherein, operably linked to a transgene. In an embodiment, a plant, planttissue, or plant cell comprises a vector comprising a gene expressioncassette, as disclosed herein. In an embodiment, a vector can be aplasmid, a cosmid, a bacterial artificial chromosome (BAC), abacteriophage, or a virus fragment.

In an embodiment, a plant, plant tissue, or plant cell according to themethods disclosed herein can be monocotyledonous. The monocotyledonplant, plant tissue, or plant cell can be, but not limited to, corn,rice, wheat, sugarcane, barley, rye, sorghum, orchids, bamboo, banana,cattails, lilies, oat, onion, millet, and triticale.

In an embodiment, a plant, plant tissue, or plant cell according to themethods disclosed herein can be dicotyledonous. The dicotyledon plant,plant tissue, or plant cell can be, but is not limited to, rapeseed,canola, Indian mustard, Ethiopian mustard, soybean, sunflower, andcotton.

With regard to the production of genetically modified plants, methodsfor the genetic engineering of plants are well known in the art. Forinstance, numerous methods for plant transformation have been developed,including biological and physical transformation protocols fordicotyledonous plants as well as monocotyledonous plants (e.g.,Goto-Fumiyuki et al., Nature Biotech 17:282-286 (1999); Mild et al.,Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. andThompson, J. E. Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993)). Inaddition, vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available, for example, inGruber et al., Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds., CRC Press, Inc., Boca Raton, pp.89-119 (1993).

One of skill in the art will recognize that after the exogenous sequenceis stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection can be performed by growing the engineered plantmaterial on media containing an inhibitory amount of the antibiotic orherbicide to which the transforming gene construct confers resistance.Further, transformed cells can also be identified by screening for theactivities of any visible marker genes (e.g., the yfp, gfp,□-glucuronidase, luciferase, B or Cl genes) that may be present on therecombinant nucleic acid constructs. Such selection and screeningmethodologies are well known to those skilled in the art.

Physical and biochemical methods also may be used to identify plant orplant cell transformants containing inserted gene constructs. In certainembodiments, the disclosure relates to a method that includes confirminga modification of genomic DNA such as the a gene expression cassetteinserted into the genome of plants. In certain embodiments, the methodof confirming such a modification of the genome includes confirmation bya PCR based assay, Southern blot assay, Northern blot assay, proteinexpression assay, Western blot assay, ELISA assay, or Next GenerationSequencing assay.

Accordingly, a modification of genomic DNA such as a gene expressioncassette inserted into the genome of plants can be confirmed in avariety of ways, including using a primer or probe of the sequence. Incertain embodiments, the stably integrated transgene may be detectedbased on the constitutive or selective expression of the transgene insome tissues of the plant or at some developmental stages, or thetransgene may be expressed in substantially all plant tissues,substantially along its entire life cycle.

Confirmation of a gene expression cassette inserted into the genome ofplants may be carried out by any suitable method of amplification. Seegenerally, Kwoh et al., Am. Biotechnol. Lab. 8, 14-25 (1990). Examplesof suitable amplification techniques include, but are not limited to,polymerase chain reaction, ligase chain reaction, strand displacementamplification (see generally G. Walker et al., Proc. Natl. Acad. Sci.USA 89, 392-396 (1992); G. Walker et al., Nucleic Acids Res. 20,1691-1696 (1992)), transcription-based amplification (see D. Kwoh etal., Proc. Natl. Acad Sci. USA 86, 1173-1177 (1989)), self-sustainedsequence replication (or “35R”) (see J. Guatelli et al., Proc. Natl.Acad. Sci. USA 87, 1874-1878 (1990)), the Q0 replicase system (see P.Lizardi et al., BioTechnology 6, 1197-1202 (1988)), nucleic acidsequence-based amplification (or “NASBA”) (see R. Lewis, GeneticEngineering News 12 (9), 1 (1992)), the repair chain reaction (or “RCR”)(see R. Lewis, supra), and boomerang DNA amplification (or “BDA”) (seeR. Lewis, supra). Polymerase chain reaction is generally preferred.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity.

As used herein, the term “polymerase chain reaction” and “PCR” generallyrefers to the method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification (U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188; hereinincorporated by reference). This process for amplifying the targetsequence comprises introducing an excess of two oligonucleotide primersto the DNA mixture containing the desired target sequence, followed by aprecise sequence of thermal cycling in the presence of a DNA polymerase.The two primers are complementary to their respective strands of thedouble stranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and, therefore, this length is acontrollable parameter. By virtue of the repeating aspect of theprocess, the method is referred to as the “polymerase chain reaction”(hereinafter “PCR”). Because the desired amplified segments of thetarget sequence become the predominant sequences (in tams ofconcentration) in the mixture, they are said to be “PCR amplified.

The term “reverse-transcriptase” or “RT-PCR” refers to a type of PCRwhere the starting material is mRNA. The starting mRNA is enzymaticallyconverted to complementary DNA or “cDNA” using a reverse transcriptaseenzyme. The cDNA is then used as a “template” for a “PCR” reaction.

In an embodiment, the amplification reaction is quantified. In otherembodiments, the amplification reaction is quantitated using a signatureprofile, in which the signature profile is selected from the groupconsisting of a melting temperature or a fluorescence signature profile.

The nucleic acid molecule of embodiments of the disclosure, or segmentsthereof, can be used as primers for PCR amplification. In performing PCRamplification, a certain degree of mismatch can be tolerated betweenprimer and template. Therefore, mutations, deletions, and insertions(especially additions of nucleotides to the 5′ or 3′ end) of theexemplified primers fall within the scope of the subject disclosure.Mutations, insertions, and deletions can be produced in a given primerby methods known to an ordinarily skilled artisan.

Molecular Beacons have been described for use in sequence detection.Briefly, a FRET oligonucleotide probe is designed that overlaps theflanking genomic and insert DNA junction. The unique structure of theFRET probe results in it containing a secondary structure that keeps thefluorescent and quenching moieties in close proximity. The FRET probeand PCR primers (one primer in the insert DNA sequence and one in theflanking genomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe(s) to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal indicates thepresence of the flanking genomic/transgene insert sequence due tosuccessful amplification and hybridization. Such a molecular beaconassay for detection of as an amplification reaction is an embodiment ofthe subject disclosure.

Hydrolysis probe assay, otherwise known as TAQMAN® (Life Technologies,Foster City, Calif.), is a method of detecting and quantifying thepresence of a DNA sequence. Briefly, a FRET oligonucleotide probe isdesigned with one oligo within the transgene and one in the flankinggenomic sequence for event-specific detection. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Hybridization of the FRET probe results incleavage and release of the fluorescent moiety away from the quenchingmoiety on the FRET probe. A fluorescent signal indicates the presence ofthe flanking/transgene insert sequence due to successful amplificationand hybridization. Such a hydrolysis probe assay for detection of as anamplification reaction is an embodiment of the subject disclosure.

KASPar assays are a method of detecting and quantifying the presence ofa DNA sequence. Briefly, the genomic DNA sample comprising the a geneexpression cassette inserted into the genome of plants is screened usinga polymerase chain reaction (PCR) based assay known as a KASPAR® assaysystem. The KASPAR® assay used in the practice of the subject disclosurecan utilize a KASPAR® PCR assay mixture which contains multiple primers.The primers used in the PCR assay mixture can comprise at least oneforward primers and at least one reverse primer. The forward primercontains a sequence corresponding to a specific region of the donor DNApolynucleotide, and the reverse primer contains a sequence correspondingto a specific region of the genomic sequence. In addition, the primersused in the PCR assay mixture can comprise at least one forward primersand at least one reverse primer. For example, the KASPAR® PCR assaymixture can use two forward primers corresponding to two differentalleles and one reverse primer. One of the forward primers contains asequence corresponding to specific region of the endogenous genomicsequence. The second forward primer contains a sequence corresponding toa specific region of the donor DNA polynucleotide. The reverse primercontains a sequence corresponding to a specific region of the genomicsequence. Such a KASPAR® assay for detection of an amplificationreaction is an embodiment of the subject disclosure.

In some embodiments the fluorescent signal or fluorescent dye isselected from the group consisting of a HEX fluorescent dye, a FAMfluorescent dye, a JOE fluorescent dye, a TET fluorescent dye, a Cy 3fluorescent dye, a Cy 3.5 fluorescent dye, a Cy 5 fluorescent dye, a Cy5.5 fluorescent dye, a Cy 7 fluorescent dye, and a ROX fluorescent dye.

In other embodiments the amplification reaction is run using suitablesecond fluorescent DNA dyes that are capable of staining cellular DNA ata concentration range detectable by flow cytometry, and have afluorescent emission spectrum which is detectable by a real timethermocycler. It should be appreciated by those of ordinary skill in theart that other nucleic acid dyes are known and are continually beingidentified. Any suitable nucleic acid dye with appropriate excitationand emission spectra can be employed, such as YO-PRO-1®, SYTOX GREEN®,SYBR GREEN I®, SYTO11®, SYTO12®, SYTO13®, BOBO®, YOYO®, and TOTO®. Inone embodiment, a second fluorescent DNA dye is SYTO13® used at lessthan 10 μM, less than 4 μM, or less than 2.7 μM.

In further embodiments, Next Generation Sequencing (NGS) can be used forconfirming a gene expression cassette inserted into the genome ofplants. As described by Brautigma et al., 2010, DNA sequence analysiscan be used to determine the nucleotide sequence of the isolated andamplified fragment. The amplified fragments can be isolated andsub-cloned into a vector and sequenced using chain-terminator method(also referred to as Sanger sequencing) or Dye-terminator sequencing. Inaddition, the amplicon can be sequenced with Next Generation Sequencing.NGS technologies do not require the sub-cloning step, and multiplesequencing reads can be completed in a single reaction. Three NGSplatforms are commercially available, the Genome Sequencer FLX from 454Life Sciences/Roche, the Illumina Genome Analyser from Solexa andApplied Biosystems' SOLiD (acronym for: “Sequencing by Oligo Ligationand Detection”). In addition, there are two single molecule sequencingmethods that are currently being developed. These include the trueSingle Molecule Sequencing (ISMS) from Helicos Bioscience and the SingleMolecule Real Time sequencing (SMRT) from Pacific Biosciences.

The Genome Sequencher FLX which is marketed by 454 Life Sciences/Rocheis a long read NGS, which uses emulsion PCR and pyrosequencing togenerate sequencing reads. DNA fragments of 300-800 bp or librariescontaining fragments of 3-20 kbp can be used. The reactions can produceover a million reads of about 250 to 400 bases per run for a total yieldof 250 to 400 megabases. This technology produces the longest reads butthe total sequence output per run is low compared to other NGStechnologies.

The Illumina Genome Analyser which is marketed by Solexa is a short readNGS which uses sequencing by synthesis approach with fluorescentdye-labeled reversible terminator nucleotides and is based onsolid-phase bridge PCR. Construction of paired end sequencing librariescontaining DNA fragments of up to 10 kb can be used. The reactionsproduce over 100 million short reads that are 35-76 bases in length.This data can produce from 3-6 gigabases per run.

The Sequencing by Oligo Ligation and Detection (SOLiD) system marketedby Applied Biosystems is a short read technology. This NGS technologyuses fragmented double stranded DNA that are up to 10 kbp in length. Thesystem uses sequencing by ligation of dye-labelled oligonucleotideprimers and emulsion PCR to generate one billion short reads that resultin a total sequence output of up to 30 gigabases per run.

tSMS of Helicos Bioscience and SMRT of Pacific Biosciences apply adifferent approach which uses single DNA molecules for the sequencereactions. The tSMS Helicos system produces up to 800 million shortreads that result in 21 gigabases per run. These reactions are completedusing fluorescent dye-labelled virtual terminator nucleotides that isdescribed as a “sequencing by synthesis” approach.

The SMRT Next Generation Sequencing system marketed by PacificBiosciences uses a real time sequencing by synthesis. This technologycan produce reads of up to 1000 bp in length as a result of not beinglimited by reversible terminators. Raw read throughput that isequivalent to one-fold coverage of a diploid human genome can beproduced per day using this technology.

In another embodiment, the confirmation of a gene expression cassetteinserted into the genome of plants can be completed using blottingassays, including Western blots, Northern blots, and Southern blots.Such blotting assays are commonly used techniques in biological researchfor the identification and quantification of biological samples. Theseassays include first separating the sample components in gels by anelectrophoretic method, followed by transfer of the electrophoreticallyseparated components from the gels to transfer membranes that are madeof materials such as nitrocellulose, polyvinylidene fluoride (PVDF), orNylon. Analytes can also be directly spotted on these supports ordirected to specific regions on the supports by applying vacuum,capillary action, or pressure, without prior separation. The transfermembranes are then commonly subjected to a post-transfer treatment toenhance the ability of the analytes to be distinguished from each otherand detected, either visually or by automated readers.

In a further embodiment the confirmation of a gene expression cassetteinserted into the genome of plants can be completed using an ELISAassay, which uses a solid-phase enzyme immunoassay to detect thepresence of a substance, usually an antigen, in a liquid sample or wetsample. Antigens from the sample are attached to a surface of a plate.Then, a further specific antibody is applied over the surface so it canbind to the antigen. This antibody is linked to an enzyme, and, in thefinal step, a substance containing the enzyme's substrate is added. Thesubsequent reaction produces a detectable signal, most commonly a colorchange in the substrate.

The present disclosure also encompasses seeds of the transgenic plantsdescribed above wherein the seed comprises the transgene or geneexpression cassette. The present disclosure further encompasses theprogeny, clones, cell lines or cells of the transgenic plants describedabove wherein said progeny, clone, cell line or cell comprise thetransgene or gene construct.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

EXAMPLES Example 1: Identification of Regulatory Elements from Brassicanapus

Brassica napus gene regulatory elements were identified via a microarrayprofiling approach. The regulatory elements were then isolated andcloned to characterize the expression profile of the regulatory elementsfor use in transgenic plants. Transgenic Arabidopsis lines stablytransformed with a Phiyfp gene and a pat selectable marker gene wereproduced, and the transgene expression levels and tissue specificity wasassessed. As such, Brassica napus regulatory elements were identifiedand characterized. Disclosed for the first time are promoter and 3′-UTRregulatory elements for use in gene expression constructs.

Microarray Profiling Approach

Developing Brassica napus seeds were collected from both a transgenichomozygote line and untransformed wildtype plants at 15, 20, 25, 30, 35and 42 days after pollination (DAP). Next, the seeds were analyzed via asingle-color, global gene expression profiling design to determineglobal levels of gene expression for each of the defined time points.Three identical replicates of individual 60-mer oligonucleotide arrays(Agilent Technologies Inc., Santa Clara, Calif.) were hybridized withamplified, Cy3 labeled cRNA from each sample. A custom designed 60-mercomprehensive transcriptome-wide canola oligonucleotide array (eArray,Agilent Technologies, Inc., Santa Clara, Calif.) was used to carry outthe hybridizations. The oligonucleotide array contains more than 37,000different canola transcripts obtained from public data sources (AgilentTechnologies, Inc., Santa Clara, Calif.).

The 60-mer oligonucleotides were synthesized in-situ using theSURE-PRINT™ technology from the manufacturer (Agilent Technologies,Inc., Santa Clara, Calif.). To efficiently measure the expression levelsof each transcript, the oligonucleotides present in the array weredesigned to be unique and specific for each target to efficientlyhybridize with the predicted target sequence. Oligonucleotides thatformed a duplex with more than one transcript were eliminated from thearray. Each oligonucleotide also fulfilled the chemical and physicalproperties required for optimal performance throughout microarrayprocessing. In addition, specific and unique oligonucleotidesrepresenting the newly introduced genes as well as several other genesof interest were also included in the custom designed canolaoligonucleotide array. These criteria were used to produce the customdesigned 60-mer comprehensive transcriptome-wide canola oligonucleotidearray.

Samples of developing seeds were obtained at 15, 20, 25, 30, 35 and 42days after pollination (DAP) from multiple plants of each genotype(NEX710® wildtype and AnD9DS transgenic lines). The seeds were frozenand pooled to be used as starting material for RNA isolation andpurification. For labeling, a total of 1.0 μg of purified total RNA fromeach sample was reverse transcribed, amplified and labeled with Cy3-CTPfollowing the Agilent One-Color Microarray-Based Gene ExpressionQuickAmp Labeling Protocol™ (Agilent, Santa Clara, Calif.).Oligonucleotide gene expression arrays were hybridized using the AgilentTechnologies Gene Expression Hybridization Kit™ and WASH BUFFER KIT™(Agilent, Santa Clara, Calif.). Hybridizations were carried out on afully automated TECAN H54800 PRO™ (TECAN, Research Triangle Park, N.C.)hybridization station.

After scanning and feature extraction, raw data files were uploaded intoGeneSpring GX version 10.0.2™ (Agilent Technologies, Santa Clara,Calif.). Quality control on samples based on spike-in controls wasperformed to ensure that the generated data was of sufficient qualitybefore generating a report by GENESPRING®. Next, the resulting data wasnormalized using a global percentile shift normalization method tominimize systematic non-biological differences and standardize arraysfor cross comparisons. The normalized data was then filtered byselecting entities that were flagged as “Present” in every single sampleunder study, and eliminating entities flagged as “Marginal” or “Absent.”The normalized and filtered list of entities was used as input forstatistical analysis using a two-way ANOVA method with a correctedp-value cut-off of p<0.05 defining DAP and genotype as parameters. Theglobal gene expression profile of Brassica napus seed development wasdefined for all time points in the study. An additional set of selectioncriteria was applied to identify genes that consistently expressed athigh levels (>50,000 pixels/spot) in all samples during early Brassicanapus seed development.

Additional genes were manually selected based upon gene annotation tobring the total candidate pool to 88 targets. To refine this pool, theexpression level was verified against known oils biosynthetic geneexpression levels with quantitative Real Time Polymerase Chain Reaction(RT-qPCR). RNA from the 15 and 20 DAP timepoints was examined, as wastotal RNA extracted and purified from young canola leaves. cDNAsynthesis for RT-qPCR was conducted with SUPERSCRIPTIII™ (Invitrogen,Carlsbad, Calif.). Real Time PCR reactions were carried out on aLIGHTCYCLER® 480 instrument (Roche, Indianapolis, Ind.) using AbGeneAbsolute Blue SYBR green master Mix™ (Thermo Fisher). Primers forRT-qPCR were designed using PRIMER3™ (MIT, Cambridge, Mass.). Primerswere designed to an optimal Tm of 60 C. Amplicon sizes ranged from100-224 bp. Primers were selected to produce an amplicon in the 3′region of the transcribed target sequence. For normalization purposes, 4endogenous oils biosynthetic genes were also measured, including BnACP05(acyl-carrier protein), BnKCS (ketoacyl-CoA synthase), BnKASIII(ketoacyl-ACP synthase), and BnSAD (stearoyl-ACP desaturase).

To further filter the candidate pool, genes which displayed expressionin early seeds higher than that of ACP05 (GenBank: X16114.1) wereselected. Genes also were required to exhibit expression increases fromleaf to early seed that were greater than the seed/leaf expressiondifferential of KASII (GenBank: AF244520.1). These filters reduced thelist of candidates down to a specific gene for identification of atarget promoter. Accordingly, the microarray assay was used to identifypromoter and 3′UTR gene regulatory elements from Brassica napus thathighly expressed cDNA at 15 DAP, and was preferentially expressed inseed.

Example 2: Gene Regulatory Element Identification

A specific sequence was selected from the Brassica napus microarraygenerated data using the screening parameters described above. A PCRreaction was used to isolate the specific promoter and 3′UTR sequencesfrom the Brassica napus c.v. Nex710. The PCR primers were designed fromthe expressed sequence tag contig 27160, assembled from publiclyavailable ESTs Genbank: EV001081.1, CD813186.1, ES989902.1 andEV088583.1), and the genomic contig ctg7180009837416 from Brassicaoleracea c.v. TO1000, identified as having homology to the EST contig27160. The extracted promoter and 3′ UTR Brassica napus regulatorysequences were obtained and further characterized via DNA sequencing.The promoter sequence of the Brassica napus gene labeled as GALE1, fromthe Brassica napus c.v. Nex710 genome is provided as a 1429 bp promotersequence of SEQ ID NO:1. The 500 bp 3′-UTR sequence is provided below asSEQ ID NO:2.

SEQ ID NO: 1 caacaaaaatgcactttttcgccaaaaatacatttttcttcaaaaaccgcaaaaatattttctgccaaacccgtaaaaatactatttttctgccgaaacgtaaaaaaaaatattttaattattttattaacaagtccacttggatgtagatgaaaatttaaaaaatgaaaagcaaacgaacatagttgcattcagatgattcatctggatgcatggacgaaatgaagaaacgaacaacacccatatagagcatctggataagacatctagatggatcattacaaaagaacagggcctaaacatgtgagatgtttgaagcaatcagtcaaaagtaaccaccaaatcgaattatgaaagcgttgattggatggacaagtttaacaaccattgtttgattggacaacgccgttatctaaacttttagtgtgctgtgtacatcattactatgaatcagttagttaaaaatattatggtcagtgaatgacagtaagattacttcagaacttgagagatttaccgcaaaaagaaacacaataacgcgtaggaaaaatatcctctgttttttgcaattattctcgtagatttggttatcagtaggtatcacgttttacaaaaatagaattacaatacatgccgcaagaaaaagactttctctttttaatttccccaatttggttatcagtattcagtaagtttcacatttttacaaaaatataaattaaaatacatactgcaagaaaaatacttttttaatttcgccaatttggttatcagtagttttcacatttttacaaaaatataattaaaatataaactgcaacaaaaagacttatctttttaatttccccaatttggttatcagtattcagtaggtttcacatttacaaaaatattattaaaatacatactgcaagaaacatacctttttaatttcgccaatctggttatcagtagttttcacatttttacaaaaatagaattaaaatacaaactgcaacaaaaagacttatctttttaatttccaccaataagttatttatttatttaatcctcccgtgaggaaaaagacaagattgaggatgaatatacgtaactgaaaattgaggaaacagagccatcaacctttcaacacggatgatcatcatcatcactctctgccgcctttaaatagaaaccaacaaagacattcttgagcccacactcactcctttcctatttcttcgctttgcgtgccttccttccttcttatctacttgtatcccacaaaaagctacttaataccatttaataaagaccccaactttcttgtgtcttctctcttatcatcttcgctgtgatctctctgtctccctctctcttatccaaaagattagtataaaaggatcgatctttccttgtgggttcttccataaaacttcgattctcgact SEQ ID NO: 2Actttactctttctctctaatcgctcaatatacaaaagaaaagtgtttacatacacacatcatatatagtttgcttttagtttccatgtaaccgaacgggtctgtttacttctatgaataaaatagctagttgatgattctgttgattgatacactctatggatagttcaagattttattacaatccaacgatgatttgtatcaaatagagcccaccagatcaagaaagcatactccagaagcttttgttcaatctaccatcagataacatatcaataaccatcttcatggtggaaccatctgcagcaaacccacacctcttcatttcttctatgagttcaactgaagcgactacaccactacctccgagatgaactcggatcagtgtgttgtatgtacactcatttggcgcaatcccatcctcctctcccatctttttaaacaacatatccgcttcagacagtgagcctttcttacacagtcctgcaatcattatggta

Example 3: Brassica napus Promoter and 3′UTR Construct

A gene expression cassette was constructed that was comprised of thefull-length Brassica napus GALE1 gene promoter of SEQ ID NO:1, yellowfluorescent protein gene (Phiyfp; Shagin et al., (2004) Mol Biol Evol21; 841-50) which contains the Solanum tuberosum, light specific tissueinducible LS-1 gene (ST-LS1 intron; Genbank Acc No. X04753), and theBrassica napus GALE13′UTR of SEQ ID NO:2 using standard recombinant DNAtechniques. This gene expression cassette was flanked by att sites.Next, a GATEWAY® LR CLONASE II® (Life Technologies, Carlsbad, Calif.)reaction was performed with the resulting entry plasmid containing theyfp gene expression cassette, under the control of the Brassica napusGALE1 gene promoter and terminated by the Brassica napus GALE1 gene 3′UTR, and a destination vector leading to a final expression vector,pDAB113903. The destination vector contained a selectable markercassette comprised of a pat gene (Wohlleben et al., Gene 70:25-37; 1988)driven by the Cassava vein mosaic virus promoter (CsVMV promoter;Verdaguer et al., Plant Molecular Biology 31:1129-1139; 1996) andterminated by an Agrobacterium tumefaciens open reading frame 13′-UTR(AtuORF13′UTR; Huang et al., J. Bacteriol. 172:1814-1822; 1990). Theresulting construct, pDAB113903 is a heterologous expression constructthat contains an yfp gene expression cassette (SEQ ID NO:3) and a patgene expression construct (SEQ ID NO:4) is presented as a plasmid map inFIG. 1.

(provides the nucleic acid sequence for theyellow fluorescent protein gene expression cassette from pDAB113903)SEQ ID NO: 3 caacaaaaatgcactttttcgccaaaaatacatttttcttcaaaaaccgcaaaaatattttctgccaaacccgtaaaaatactatttttctgccgaaacgtaaaaaaaaatattttaattattttattaacaagtccacttggatgtagatgaaaatttaaaaaatgaaaagcaaacgaacatagttgcattcagatgattcatctggatgcatggacgaaatgaagaaacgaacaacacccatatagagcatctggataagacatctagatggatcattacaaaagaacagggcctaaacatgtgagatgtttgaagcaatcagtcaaaagtaaccaccaaatcgaattatgaaagcgttgattggatggacaagtttaacaaccattgtttgattggacaacgccgttatctaaacttttagtgtgctgtgtacatcattactatgaatcagttagttaaaaatattatggtcagtgaatgacagtaagattacttcagaacttgagagatttaccgcaaaaagaaacacaataacgcgtaggaaaaatatcctctgttttttgcaattattctcgtagatttggttatcagtaggtatcacgttttacaaaaatagaattacaatacatgccgcaagaaaaagactttctctttttaatttccccaatttggttatcagtattcagtaagtttcacatttttacaaaaatataaattaaaatacatactgcaagaaaaatacttttttaatttcgccaatttggttatcagtagttttcacatttttacaaaaatataattaaaatataaactgcaacaaaaagacttatctttttaatttccccaatttggttatcagtattcagtaggtttcacatttacaaaaatattattaaaatacatactgcaagaaacatacctttttaatttcgccaatctggttatcagtagttttcacatttttacaaaaatagaattaaaatacaaactgcaacaaaaagacttatctttttaatttccaccaataagttatttatttatttaatcctcccgtgaggaaaaagacaagattgaggatgaatatacgtaactgaaaattgaggaaacagagccatcaacctttcaacacggatgatcatcatcatcactctctgccgcctttaaatagaaaccaacaaagacattcttgagcccacactcactcctttcctatttcttcgctttgcgtgccttccttccttcttatctacttgtatcccacaaaaagctacttaataccatttaataaagaccccaactttcttgtgtcttctctcttatcatcttcgctgtgatctctctgtctccctctctcttatccaaaagattagtataaaaggatcgatctttccttgtgggttcttccataaaacttcgattctcgactggatctccatgtcatctggagcacttctctttcatgggaagattccttacgttgtggagatggaagggaatgttgatggccacacctttagcatacgtgggaaaggctacggagatgcctcagtgggaaaggtatgtttctgcttctacctttgatatatatataataattatcactaattagtagtaatatagtatttcaagtatttttttcaaaataaaagaatgtagtatatagctattgcttttctgtagtttataagtgtgtatattttaatttataacttttctaatatatgaccaaaacatggtgatgtgcaggttgatgcacaattcatctgtactaccggagatgttcctgtgccttggagcacacttgtcaccactctcacctatggagcacagtgctttgccaagtatggtccagagttgaaggacttctacaagtcctgtatgccagatggctatgtgcaagagcgcacaatcacctttgaaggagatggcaacttcaagactagggctgaagtcacctttgagaatgggtctgtctacaatagggtcaaactcaatggtcaaggcttcaagaaagatggtcacgtgttgggaaagaacttggagttcaacttcactccccactgcctctacatctggggagaccaagccaaccacggtctcaagtcagccttcaagatatgtcatgagattactggcagcaaaggcgacttcatagtggctgaccacacccagatgaacactcccattggtggaggtccagttcatgttccagagtatcatcatatgtcttaccatgtgaaactttccaaagatgtgacagaccacagagacaacatgagcttgaaagaaactgtcagagctgttgactgtcgcaagacctacctttgagtagttagcttaatcacctagagctcggtcaccactttactctttctctctaatcgctcaatatacaaaagaaaagtgtttacatacacacatcatatatagtttgcttttagtttccatgtaaccgaacgggtctgtttacttctatgaataaaatagctagttgatgattctgttgattgatacactctatggatagttcaagattttattacaatccaacgatgatttgtatcaaatagagcccaccagatcaagaaagcatactccagaagcttttgttcaatctaccatcagataacatatcaataaccatcttcatggtggaaccatctgcagcaaacccacacctcttcatttcttctatgagttcaactgaagcgactacaccactacctccgagatgaactcggatcagtgtgttgtatgtacactcatttggcgcaatcccatcctcctctcccatctttttaaacaacatatccgcttcagacagtgagcctttcttacacagtcctg caatcattatggta.(provides the nucleic acid sequence for thephosphinothricin acetyl transferase geneexpression cassette from pDAB113903) SEQ ID NO: 4ccagaaggtaattatccaagatgtagcatcaagaatccaatgtttacgggaaaaactatggaagtattatgtaagctcagcaagaagcagatcaatatgcggcacatatgcaacctatgttcaaaaatgaagaatgtacagatacaagatcctatactgccagaatacgaagaagaatacgtagaaattgaaaaagaagaaccaggcgaagaaaagaatcttgaagacgtaagcactgacgacaacaatgaaaagaagaagataaggtcggtgattgtgaaagagacatagaggacacatgtaaggtggaaaatgtaagggcggaaagtaaccttatcacaaaggaatcttatcccccactacttatccttttatatttttccgtgtcatttttgcccttgagttttcctatataaggaaccaagttcggcatttgtgaaaacaagaaaaaatttggtgtaagctattttctttgaagtactgaggatacaacttcagagaaatttgtaagtttgtaggtaccagatctggatcccaaaccatgtctccggagaggagaccagttgagattaggccagctacagcagctgatatggccgcggtttgtgatatcgttaaccattacattgagacgtctacagtgaactttaggacagagccacaaacaccacaagagtggattgatgatctagagaggttgcaagatagatacccttggttggttgctgaggttgagggtgttgtggctggtattgcttacgctgggccctggaaggctaggaacgcttacgattggacagttgagagtactgtttacgtgtcacataggcatcaaaggttgggcctaggatctacattgtacacacatttgcttaagtctatggaggcgcaaggttttaagtctgtggttgctgttataggccttccaaacgatccatctgttaggttgcatgaggctttgggatacacagcccggggtacattgcgcgcagctggatacaagcatggtggatggcatgatgttggtttttggcaaagggattttgagttgccagctcctccaaggccagttaggccagttacccaaatctgagtagttagcttaatcacctagagctcgatcggcggcaatagcttcttagcgccatcccgggttgatcctatctgtgttgaaatagttgcggtgggcaaggctctctttcagaaagacaggcggccaaaggaacccaaggtgaggtgggctatggctctcagttccttgtggaagcgcttggtctaaggtgcagaggtgttagcgggatgaagcaaaagtgtccgattgtaacaagatatgttgatcctacgtaaggatattaaagtatgtattcatcactaatataatcagtgtattccaatatgtactacgatttccaatgtctttattgtcgccgtatgtaatcggcgtcacaaaataatccccggtgactttcttttaatccaggatgaaataatatgttattataatttttgcgatttggtccgttataggaattgaagtgtgcttgaggtcggtcgccaccactcccatttcataattttacatgtatttgaaaaataaaaatttatggtattcaatttaaacacgtatacttgtaaagaatgatatcttgaaagaaatatagtttaaatatttattgataaaataacaagtcaggtattatagtccaagcaaaaacataaatttattgatgcaagtttaaattcagaaatatttcaataactgattatatcagctggtacattgccgtagatgaaagactgagtgcgatattatggtgtaatacatagg.

A positive control construct, pDAB9381, was assembled containing ayellow fluorescent protein (yfp) gene expression cassette, and aphosphinothricin acetyltransferase gene expression cassette.Specifically, the yellow fluorescent protein gene expression cassettecontains the Arabidopsis thaliana Ubiquitin 10 gene promoter (At Ubi10promoter; Callis et al., 1990, J Biol Chem 265:12486-12493), yellowfluorescence protein coding sequence (PhiYFP; Shagin et al., 2004Molecular Biology and Evolution, 21(5), 841-850) which contains theSolanum tuberosum, light specific tissue inducible LS-1 gene (ST-LS1intron; Genbank Acc No. X04753), and is terminated with theAgrobacterium tumefaciens Open Reading Frame 23 3′ Untranslated Region(AtuORF23 3′UTR). The selectable marker gene expression cassettecontains the Cassava vein Mosaic Virus Promoter (CsVMV promoter;Verdaguer et al., Plant Molecular Biology 31:1129-1139; 1996),phosphinothricin acetyl transferase (PAT; Wohlleben et al., Gene70:25-37; 1988) and Agrobacterium tumefaciens ORF13′ untranslated region(AtuORF13′ UTR; Huang et al., J. Bacteriol. 172:1814-1822; 1990). Theresulting construct, pDAB9381 is a heterologous expression constructthat contains an yfp gene expression cassette (SEQ ID NO:5) and a patgene expression construct (SEQ ID NO:5) is presented as FIG. 2.

(provides the nucleic acid sequence for theyellow fluorescent protein gene expression cassette from pDAB9381)SEQ ID NO: 5 gtcgacctgcaggtcaacggatcaggatattcttgtttaagatgttgaactctatggaggtttgtatgaactgatgatctaggaccggataagttcccttcttcatagcgaacttattcaaagaatgttttgtgtatcattcttgttacattgttattaatgaaaaaatattattggtcattggactgaacacgagtgttaaatatggaccaggccccaaataagatccattgatatatgaattaaataacaagaataaatcgagtcaccaaaccacttgccttttttaacgagacttgttcaccaacttgatacaaaagtcattatcctatgcaaatcaataatcatacaaaaatatccaataacactaaaaaattaaaagaaatggataatttcacaatatgttatacgataaagaagttacttttccaagaaattcactgattttataagcccacttgcattagataaatggcaaaaaaaaacaaaaaggaaaagaaataaagcacgaagaattctagaaaatacgaaatacgcttcaatgcagtgggacccacggttcaattattgccaattttcagctccaccgtatatttaaaaaataaaacgataatgctaaaaaaatataaatcgtaacgatcgttaaatctcaacggctggatcttatgacgaccgttagaaattgtggttgtcgacgagtcagtaataaacggcgtcaaagtggttgcagccggcacacacgagtcgtgtttatcaactcaaagcacaaatacttttcctcaacctaaaaataaggcaattagccaaaaacaactttgcgtgtaaacaacgctcaatacacgtgtcattttattattagctattgcttcaccgccttagctttctcgtgacctagtcgtcctcgtcttttcttcttcttcttctataaaacaatacccaaagcttcttcttcacaattcagatttcaatttctcaaaatcttaaaaactttctctcaattctctctaccgtgatcaaggtaaatttctgtgttccttattctctcaaaatcttcgattttgttttcgttcgatcccaatttcgtatatgttctttggtttagattctgttaatcttagatcgaagacgattttctgggtttgatcgttagatatcatcttaattctcgattagggtttcataaatatcatccgatttgttcaaataatttgagttttgtcgaataattactcttcgatttgtgatttctatctagatctggtgttagtttctagtttgtgcgatcgaatttgtcgattaatctgagtttttctgattaacagagatctccatgtcatctggagcacttctctttcatgggaagattccttacgttgtggagatggaagggaatgttgatggccacacctttagcatacgtgggaaaggctacggagatgcctcagtgggaaaggtatgtttctgcttctacctttgatatatatataataattatcactaattagtagtaatatagtatttcaagtatttttttcaaaataaaagaatgtagtatatagctattgcttttctgtagtttataagtgtgtatattttaatttataacttttctaatatatgaccaaaacatggtgatgtgcaggttgatgcacaattcatctgtactaccggagatgttcctgtgccttggagcacacttgtcaccactctcacctatggagcacagtgctttgccaagtatggtccagagttgaaggacttctacaagtcctgtatgccagatggctatgtgcaagagcgcacaatcacctttgaaggagatggcaacttcaagactagggctgaagtcacctttgagaatgggtctgtctacaatagggtcaaactcaatggtcaaggcttcaagaaagatggtcacgtgttgggaaagaacttggagttcaacttcactccccactgcctctacatctggggagaccaagccaaccacggtctcaagtcagccttcaagatatgtcatgagattactggcagcaaaggcgacttcatagtggctgaccacacccagatgaacactcccattggtggaggtccagttcatgttccagagtatcatcatatgtcttaccatgtgaaactttccaaagatgtgacagaccacagagacaacatgagcttgaaagaaactgtcagagctgttgactgtcgcaagacctacctttgagtagttagcttaatcacctagagctcggtcaccagcataatttttattaatgtactaaattactgttttgttaaatgcaattttgctttctcgggattttaatatcaaaatctatttagaaatacacaatattttgttgcaggcttgctggagaatcgatctgctatcataaaaattacaaaaaaattttatttgcctcaattattttaggattggtattaaggacgcttaaattatttgtcgggtcactacgcatcattgtgattgagaagatcagcgatacgaaatattcgtagtactatcgataatttatttgaaaattcataagaaaagcaaacgttacatgaattgatgaaacaatacaaagacagataaagccacgcacatttaggatattggccgagattactgaatattgagtaagatcacggaatttctgacaggagcatgtcttcaattcagcccaaatggcagttgaaatactcaaaccgccccatatgcaggagcggatcattcattgtttgtttggttgcctttgccaacatgggagtccaaggtt(provides the nucleic acid sequence for thephosphinothricin acetyl transferase geneexpression cassette from pDAB9381) SEQ ID NO: 6ccagaaggtaattatccaagatgtagcatcaagaatccaatgtttacgggaaaaactatggaagtattatgtaagctcagcaagaagcagatcaatatgcggcacatatgcaacctatgttcaaaaatgaagaatgtacagatacaagatcctatactgccagaatacgaagaagaatacgtagaaattgaaaaagaagaaccaggcgaagaaaagaatcttgaagacgtaagcactgacgacaacaatgaaaagaagaagataaggtcggtgattgtgaaagagacatagaggacacatgtaaggtggaaaatgtaagggcggaaagtaaccttatcacaaaggaatcttatcccccactacttatccttttatatttttccgtgtcatttttgcccttgagttttcctatataaggaaccaagttcggcatttgtgaaaacaagaaaaaatttggtgtaagctattttctttgaagtactgaggatacaacttcagagaaatttgtaagtttgtaggtaccagatctggatcccaaaccatgtctccggagaggagaccagttgagattaggccagctacagcagctgatatggccgcggtttgtgatatcgttaaccattacattgagacgtctacagtgaactttaggacagagccacaaacaccacaagagtggattgatgatctagagaggttgcaagatagatacccttggttggttgctgaggttgagggtgttgtggctggtattgcttacgctgggccctggaaggctaggaacgcttacgattggacagttgagagtactgtttacgtgtcacataggcatcaaaggttgggcctaggatctacattgtacacacatttgcttaagtctatggaggcgcaaggttttaagtctgtggttgctgttataggccttccaaacgatccatctgttaggttgcatgaggctttgggatacacagcccggggtacattgcgcgcagctggatacaagcatggtggatggcatgatgttggtttttggcaaagggattttgagttgccagctcctccaaggccagttaggccagttacccaaatctgagtagttagcttaatcacctagagctcgatcggcggcaatagcttcttagcgccatcccgggttgatcctatctgtgttgaaatagttgcggtgggcaaggctctctttcagaaagacaggcggccaaaggaacccaaggtgaggtgggctatggctctcagttccttgtggaagcgcttggtctaaggtgcagaggtgttagcgggatgaagcaaaagtgtccgattgtaacaagatatgttgatcctacgtaaggatattaaagtatgtattcatcactaatataatcagtgtattccaatatgtactacgatttccaatgtctttattgtcgccgtatgtaatcggcgtcacaaaataatccccggtgactttcttttaatccaggatgaaataatatgttattataatttttgcgatttggtccgttataggaattgaagtgtgcttgaggtcggtcgccaccactcccatttcataattttacatgtatttgaaaaataaaaatttatggtattcaatttaaacacgtatacttgtaaagaatgatatcttgaaagaaatatagtttaaatatttattgataaaataacaagtcaggtattatagtccaagcaaaaacataaatttattgatgcaagtttaaattcagaaatatttcaataactgattatatcagctggtacattgccgtagatgaaagactgagtgcgatattatggtgtaatacatagg

Example 4: Plant Transformation and Molecular Confirmation

Agrobacterium Preparation

Next, 60 μl of an Agrobacterium strain, in 50% glycerol (previouslyprepared and frozen at −80° C.), containing either one of the abovedescribed binary plasmids, was used to prepare a 5 ml starter culture ofYEP liquid (BACTO PEPTONE™10.0 gm/L, Yeast Extract 10.0 gm/L, and sodiumchloride 5.0 gm/L) containing spectinomycin (100 mg/L), kanamycin (50mg/L), and rifampicin (10 mg/L) and incubated for overnight at 28° C.with aeration. The Agrobacterium starter was then inoculated into 300 mLYEP liquid with spectinomycin (100 mg/L), kanamycin (50 mg/L), andrifampicin (10 mg/L) into sterile 500 mL baffled flask(s) and shaken at200 rpm at 28° C. overnight. The cultures were centrifuged at 6000 rpmand resuspended in an equal volume of ½×MS-medium containing 10% (w/v)sucrose, 10 ug/L 6-benzylaminopurine, and 0.03% Silwet L-77 prior totransformation of plant tissue.

Arabidopsis Transformation

Arabidopsis was transformed using the floral dip method adapted fromClough and Bent (1998). A validated Agrobacterium glycerol stockcontaining one of the binary plasmids described above was used toinoculate a 5 mL pre-culture of YEP broth containing spectinomycin (100mg/L), kanamycin (50 mg/L), and rifampicin (10 mg/L). The culture wasincubated overnight at 28° C. with aeration. The pre-culture was thenbulked up to 300 mL with the same antibiotic selection and incubatedagain at 28° C. with constant agitation at 225 rpm. The cells werepelleted at approximately 5,000×g for 15 minutes at 4° C., and thesupernatant discarded. The cell pellet was gently resuspended in 300 mLinoculation medium containing: 10% (w/v) sucrose, 10 ug/L6-benzylaminopurine, and 0.03% Silwet L-77. Plants at 41 days old(primary inflorescences cut back at 35 days) were inverted and dippedinto the medium. The plants (now denoted as T₀) were placed on theirsides in a transparent covered plastic tub overnight, and then setupright in the growth chamber the following day. The plants were grownat 22° C., with a 16-hour light/8-hour dark photoperiod. Four weeksafter dipping, the water was cut off and plants were allowed to dry downfor a week in preparation for T₁ seed harvesting.

T₁ seed was sown on 10.5″×21″ germination trays, each receiving a 200 mgaliquots of stratified T₁ seed (40,000 seed) that had previously beensuspended in 40 mL of 0.1% agar solution and stored at 4° C. for 2 daysto ensure synchronous seed germination (vernalization).

Sunshine Mix LP5 soil media was covered with fine vermiculite andsubirrigated with Hoagland's solution until wet, then allowed to gravitydrain. Each 40 mL aliquot of stratified seed was sown evenly onto thevermiculite with a pipette and covered with humidity domes for 4-5 days.Domes were removed 1 day prior to initial transformant selection usingglufosinate (Liberty).

Seven days after planting (DAP) and again at 9 DAP, T₁ plants (cotyledonand 2-4-1f stage, respectively) were sprayed with a 0.2% solution ofLiberty herbicide (200 g ai/L glufosinate, Bayer Crop Sciences, KansasCity, Mo.) at a spray volume of 10 ml/tray (703 L/ha) using a DeVilbisscompressed air spray tip to deliver an effective rate of 280 g ai/haglufosinate per application. Survivors (putative transformed plantsactively growing) were identified 3 days after the final spraying andtransplanted individually into 3-inch pots prepared with Sunshine MixLP5 in the greenhouse 7 days after the final spray selection (16 DAP).The transplants were reared in the greenhouse (22±5° C., 50±30% RH, 14 hlight:10 dark, minimum 500 μE/m² s¹ natural+supplemental light).Molecular analysis was completed on the surviving T₁ plants to confirmthat the pat herbicide selectable marker gene had integrated into thegenome of the plants.

Molecular Confirmation

Putative transgenic Arabidopsis plants were sampled for detection oftransgene presence using a quantitative PCR assay for pat. Total DNA wasextracted from the leaf samples, using QIAGEN® BioSprint96 Kit DNAextraction kit (Qiagen, Valencia, Calif.) as per manufacturer'sinstructions.

To detect the genes of interest, gene-specific DNA fragments wereamplified with hydrolysis (analogous to TAQMAN®) primer/probe setscontaining a Cy5-labeled fluorescent probe for the pat gene and aHEX-labeled fluorescent probe for the endogenous TafII-15 reference genecontrol (Genbank ID: NC 003075; Duarte et al., BMC Evol. Biol., 10:61).The following primers were used for the pat and endogenous TafII-15reference gene amplifications. The primer sequences were as follows:

pat Primers/Probes: Pat Forward Primer: TQPATS: (SEQ ID NO: 7)ACAAGAGTGGATTGATGATCTAGAGAGGT Pat Reverse Primer: TQPATA: (SEQ ID NO: 8)CTTTGATGCCTATGTGACACGTAAACAGT Pat Probe: (SEQ ID NO: 9)5′-/5Cy5/AGGGTGTTGTGGCTGGTATTGCTTACGCT/3BHQ_2/-3′TafII-15 Primers/Probes: Forward Primer: TafII-15 F: (SEQ ID NO: 10)GAGGATTAGGGTTTCAACGGAG Reverse Primer: TafII-15 R: (SEQ ID NO: 11)GAGAATTGAGCTGAGACGAGG TafII-15 Probe: (SEQ ID NO: 12)5′-/5HEX/AGAGAAGTTTCGACGGATTTCGGGC/3BHQ_2/-3′

Next, the qPCR reactions were carried out in a final volume of 10 μlreaction containing 5 μl of Roche LIGHTCYCLER® 480 Probes Master Mix(Roche Applied Sciences, Indianapolis, Ind.); 0.4 μl each of TQPATA,TQPATS, TafII-15 F and TafII-15 R primers from 10 μM stocks to a finalconcentration of 400 nM; 0.4 μl each of Pat Probe and TafII-15 Probefrom 5 μM stocks to a final concentration of 200 nM, 2 μl of genomic DNAdiluted 1:5 in water, and 0.5 μl water. The DNA was amplified in a RocheLIGHTCYCLER® 480 System under the following conditions: 1 cycle of 95°C. for 10 min followed by 40 cycles of the following 3-steps: 95° C. for10 seconds; 60° C. for 40 seconds and 72° C. for 1 second. The pat copynumber was determined by comparison of Target (gene ofinterest)/Reference (Invertase gene) values for unknown samples (outputby the LIGHTCYCLER® 480) to Target/Reference values of pat copy numbercontrols.

From the molecular confirmation, specific Arabidopsis transgenic eventsthat contained a single transgene insert of the above described plasmidswere identified. These plants were self-fertilized, and the resulting T₁seed was planted to monitor and assay T₁ seedlings for YFP proteinexpression in different plant tissues at different developmental stages.

Example 5: Transgenic Plant Expression Screening

The Arabidopsis T₁ transgenic events were grown into seedlings andassessed for YFP fluorescence at 15 DAP via fluorescent microscopy andvisual observation. Additional, observation of the Arabidopsis T₁transgenic events was completed a 7 weeks after planting, wherein theleaves, inflorescences and siliques were viewed via fluorescentmicroscopy and visual observation. Finally, observation of theArabidopsis T₁ transgenic events was completed a 10 weeks afterplanting, wherein the developing seedlings were viewed via fluorescentmicroscopy and visual observation. Seedlings and developing seeds wereimaged using a Leica DFC310 FX Stereoscope™ (Leica, Buffalo Grove, Ill.)with the following settings (e.g., excitation max—525 nm and emissionmax—482 nm). Leaves, inflorescences and siliques were imaged using theTYPHOON SCANNER™′ (GE Healthcare Life Sciences, Piscataway, N.J.) withthe following settings (e.g., Blue 488 nm laser, 670 BP 30 forchllorophyll, 520 BP 40 for Phiyfp, 350 PMT).

The YFP imaging of T₁ seedlings and plant tissues indicated that seedpreferential expression of the protein expressed under the control ofthe Brassica napus GALE1 gene promoter and terminated by the Brassicanapus GALE1 gene 3′ UTR was detected in single copy events. Visualobservations suggest that expression driven by these regulatory elementsis specific to early seed development, and early floral development. Assuch, the expression pattern driven by the Brassica napus GALE1regulatory elements was observed within the ovules of developinginflorescences (FIG. 3). The expression of the YFP protein tissue ismost likely localized within endosperm tissues during the development ofseed within Arabidopsis plants. The expression in the endosperm tissueis significant, as this tissue type makes up the majority of seedtissues during early seed development. Further expression of the YFPprotein was observed in the flowers, stems, leaves, and seeds oftransgenic events containing multiple copies of the yfp transgene.

Example 6: Expression Protein Quantification in Arabidopsis

Samples of the Arabidopsis plant seeds were assayed via PhiYFP ELISAseeds were collected and subjected to bead-milling. About 10 mg of seedmaterial was beat with 2 BBs (4.5 mm steel balls; Daisy; Rogers, Ark.)for 1 minute in a KLECCO™ bead mil 300 μl of extraction buffer (PBSsupplemented with 0.05° 4) Tween20 and 0.05%) bovine serum albumin wasadded. The samples were suspended with gentle tapping and rocked on aplatform shaker for 30 minutes at room temperature. The samples werethen spun down in a centrifuge at 14,000×g for 5 minutes. Thesupernatant was removed and analyzed via ELISA. Maxisorb Plates™ (ThermoFisher Scientific) were coated with an anti-YFP monoclonal antibody(Origene #TA150028) at a concentration of 1.0 μg/ml in 1×PBS. Followingovernight incubation at 4° C., plates were blocked with PBST (PBS+0.5%TWEEN®-20) with 0.5% bovine serum albumin for 2 hours at 37° C. Prior toanalysis, plates were washed 4 times in a plate washer using 350 μl ofPBST per wash. A purified protein reference antigen (Evrogen) wasdiluted in blocking buffer to 2 ng/ml and used to generate a standardcurve of serial dilutions from 2 ng/ml to 0.0313 ng/ml. Samples werediluted in blocking buffer to a starting dilution of 1:4 and diluted ata 1:4 rate 3 additional times (1:4, 1:16, 1:64, 1:256). Next, 100 μl ofall standards and sample dilutions were loaded in duplicate onto theELISA plate. Samples were incubated on the ELISA plate at roomtemperature for 1 hour. Following incubation, the plate was washed asabove. A rabbit anti-PhiYFP polyclonal antibody (Evrogen) was diluted to1 μg/ml in blocking buffer and added to the plate at 100 μl per well.The plate was incubated at room temperature for 1 hour prior to washing.An anti-rabbit horseradish peroxidase conjugated detection antibody(Pierce) was added to the plate at a 1:5000 dilution. The plate wasincubated at room temperature for 1 hour and washed as above. Next,1-Step Ultra TMB Substrate™ (Thermo Scientific) was added to the plateat 100 μl per well. As the wells with the lowest dilution of thestandard curve began to show blue color, the reaction was stopped byadding 50 μl of stop solution (0.4 N H2504). The plate was read in aplate reader (Molecular Devices) using SOFTMAX® Pro v5 (MolecularDevices) at a wavelength of 450 nm minus a 650 nm reference. The PhiYFPconcentration of test samples was calculated by linear regression of aquadratic standard curve.

The expression levels of YFP were quantitated and are provided in Table1 below. The expression of YEP by the Brassica napus GALE regulatoryelements ranged from 0.018 to 0.070 ng/mg within the Arabidopsis seedfor the transgenic events containing low copy number events (i.e., I-2copies). Furthermore, the results indicated that the average expressionof the low copy number event was about 0.047 ng/mg. Finally, expressionof YFP by the Brassica napus GALE regulatory elements was 6.341 ng/mgwithin the seed of Arabidopsis for the transgenic events containing highcopy number events (i.e., more than 2 copies),

TABLE 1 Quantitated expression of YFP in Arabidopsis seed. PAT copy YFPcopy Expression of Average Expression Seed Name number number YFP ng/mgseed ng/mg seed 113903[2]-004.sx001. 0.98 0.92 0.018 0.047 ng/mg for113903[2]-017.sx001. 0.93 0.86 0.047 single copy events113903[2]-019.sx001. 1.04 0.86 0.070 113903[2]-034.sx001. 0.98 0.840.053 113903[2]-023.sx001. 7.02 6.2 6.341 6.341 ng/mg for multiple copyevents Wt Negative 0.00 0.00 0.000 0.0000 Wt Negative 0.00 0.00 0.000

As such, Brassica napus GALE gene regulatory elements were identifiedand characterized. Disclosed for the first time are novel promoter and3′-UTR regulatory elements for use in gene expression constructs.

We claim:
 1. A method for expressing a heterologous coding sequence in a transgenic plant, the method comprising: transforming a plant cell with a gene expression cassette comprising a polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1 operably linked to the heterologous coding sequence, which is operably linked to a 3′-untranslated region; isolating the transformed plant cell comprising the gene expression cassette; regenerating the transformed plant cell into a transgenic plant; and, obtaining the transgenic plant, wherein the transgenic plant comprises the gene expression cassette comprising the polynucleotide sequence comprising SEQ ID NO:1.
 2. The method of claim 1, wherein the polynucleotide sequence comprises an intron having at least 90% sequence identity to an intron selected from the group consisting of a rice actin intron, a maize ubiquitin intron, and an Arabadiopsis thaliana ubiquitin 10 intron.
 3. The method of claim 1, wherein the polynucleotide sequence comprises a 5′-untranslated region.
 4. The method of claim 1, wherein transferring the plant cell is selected from the group consisting of an Agrobacterium-mediated transformation method, a biolistics transformation method, a silicon carbide transformation method, a protoplast transformation method, and a liposome transformation method.
 5. The method of claim 1, wherein the transgenic plant is selected from the group consisting of an Arabidopsis plant, a tobacco plant, a soybean plant, a canola plant and a cotton plant.
 6. A transgenic seed from the transgenic plant of claim
 1. 7. The method of claim 1, wherein the polynucleotide sequence comprises a sequence of nucleotides 1-1429 of SEQ ID NO:1.
 8. A method for isolating a polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1, the method comprising: identifying the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1; producing a plurality of oligonucleotide primer sequences, wherein the oligonucleotide primer sequences bind to the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1; amplifying the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1 from a DNA sample with oligonucleotide primer sequences selected from the plurality of oligonucleotide primer sequences; and, isolating the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1.
 9. The method of claim 8, wherein the isolated polynucleotide sequence comprises an intron having at least 90% sequence identity to an intron selected from the group consisting of a rice actin intron, a maize ubiquitin intron, and an Arabadiopsis thaliana ubiquitin 10 intron.
 10. The method of claim 8, wherein the isolated polynucleotide sequence comprises a 5′-untranslated region.
 11. The method of claim 8, wherein the isolated polynucleotide sequence is operably linked to a transgene.
 12. A gene expression cassette comprising a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO:1 operably linked to a transgene, wherein the transgene is operably linked to a 3′-untranslated region.
 13. The gene expression cassette of claim 12, wherein the transgene is selected from the group consisting of insecticidal resistance coding sequences, herbicide tolerance coding sequences, nitrogen use efficiency coding sequences, water use efficiency coding sequences, nutritional quality coding sequences, DNA binding coding sequences, and selectable marker coding sequences.
 14. The gene expression cassette of claim 12, wherein the 3′-untranslated region has at least 90% sequence identity to SEQ ID NO:2.
 15. A recombinant vector comprising the gene expression cassette of claim
 12. 16. A transgenic cell comprising the gene expression cassette of claim
 12. 17. A transgenic plant comprising the transgenic cell of claim
 16. 18. The transgenic plant of claim 17, wherein the dicotyledonous plant is selected from the group consisting of an Arabidopsis plant, a tobacco plant, a soybean plant, a canola plant and a cotton plant.
 19. A transgenic seed from the transgenic plant of claim
 17. 20. The method of claim 1, wherein the isolated polynucleotide sequence comprises a sequence of nucleotides 1-1429 of SEQ ID NO:1.
 21. A method for manufacturing a synthetic polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1, the method comprising: identifying the polynucleotide sequence comprising SEQ ID NO:1; isolating the polynucleotide sequence comprising SEQ ID NO:1; defining a plurality of polynucleotide sequences that comprise a sequence identity of at least 90% to SEQ ID NO:1; synthesizing a polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1; and, manufacturing a synthetic polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1.
 22. The method of claim 21, wherein the synthesizing comprises: identifying the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1; producing a plurality of oligonucleotide primer sequences, wherein the oligonucleotide primer sequences bind to the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1; ligating the plurality of oligonucleotide primer sequences to synthesize the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1.
 23. The method of claim 22, wherein the synthetic polynucleotide sequence comprises an intron having at least 90% sequence identity to an intron selected from the group consisting of a rice actin intron, a maize ubiquitin intron, and an Arabadiopsis thaliana ubiquitin 10 intron.
 24. The method of claim 21, wherein the synthetic polynucleotide sequence comprises a 5′-untranslated region.
 25. The method of claim 21, wherein the synthesized polynucleotide sequence is operably linked to a transgene.
 26. The method of claim 25, wherein the operably linked transgene encodes a polypeptide.
 27. A gene expression cassette comprising the synthesized polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1 operably linked to a transgene that is operably linked to a 3′-untranslated region.
 28. The gene expression cassette of claim 27, wherein the 3′-untranslated region has at least 90% sequence identity to SEQ ID NO:2.
 29. A recombinant vector comprising the gene expression cassette of claim
 26. 30. A transgenic cell comprising the gene expression cassette of claim
 26. 31. A transgenic plant comprising the transgenic cell of claim
 30. 32. The transgenic plant of claim 31, wherein the plant is selected from the group consisting of an Arabidopsis plant, a tobacco plant, a soybean plant, a canola plant and a cotton plant.
 33. A transgenic seed from the transgenic plant of claim
 31. 34. The method of claim 21, wherein the synthetic polynucleotide sequence comprises a sequence of nucleotides 1-1429 of SEQ ID NO:1. 